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Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



0 Publication number: 



0177 343 B1 



© 



EUROPEAN PATENT SPECIFICATION 



© Date of publication of patent specification: 22.07.92 @ Int. CI Ci2N 15/70, 01 2N 15/62, 

C07K 15/00, 01 2N 5/00 

@ Application number: 85307044.9 

@ Date of filing: 02.10.85 

The file contains technical information submitted 
after the application was filed and not included in 
this specification 



0 DNA, cell cultures and methods for the secretion of heterologous proteins and periplasmic protein 
recovery. 



ID 

CO 
CO 



@ Priority: 05.10.84 us 658342 
05.10.84 US 658339 
05.10.84 US 658095 

@ Date of publication of application: 
09.04.86 Bulletin 86/15 

© Publication of the grant of the patent: 
22.07.92 Bulletin 92/30 

@ Designated Contracting States: 

AT BE OH DE FR GB IT LI LU NL SE 



References cited: 
EP-A- 55 942 
EP-A-0111 389 
EP-A- 0114 695 
WO-A-84/03519 



EP-A- 0 022 242 
EP-A- 0112 012 
WO-A-84/00774 



Infection and immunity, vol. 42, n 1, October 
1983 (Washington, DC) R.N. Picken et al 
"Nucleotide sequence of the gene for heat- 
stable enterotoxin II of Escherichia coll", 
pages 269-275 



(3) Proprietor: GENENTECH, INC. 
460 Point San Bruno Boulevard 
South San Francisco California 94080(US) 

@ Inventor: Bochner, Barry Ronald 
2904 Lincoln Avenue 
Alameda California 94501 (US) 
Inventor: Chang, Chung-Nan 
231 Rosilte Street 
San Mateo California 94403(US) 
Inventor: Gray, Gregory Lawrence 
530 Elm court 

South San Francisco California 94080(US) 

Inventor: Heyneker, Herbert Louis 

501 Roehampton Road 

Hillsborough California 94010(US) 

Inventor: McFarland, Nancy Chadwick 

2019 Whiteoak Way 

San Carlos California 94070(US) 

Inventor: Olson, Kenneth Charles 

3036 Hillside Drive 

Burtingame California 9401 0(US) 



O Note: Within nine months from the publication of the mention of the grant of the European patent, any person 
may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition 

IXJ shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee 
has been paid (Art. 99(1) European patent convention). 



Rank Xerox (UK) Business Services 



EP 0 177 343 B1 



Journal of Bacteriology, vol. 154, n 1, April 
1983 (Washington DC) S. MIchaells et ai 
"Mutations that alter the signal sequence of 
alklne phosphatase in Escherichia coll", 
pages366-374 

Journal of Bacteriology, vol. 149, n 2, Feb- 
ruary 1982 (Washington DC) H. Inouye et ai 
"Signal sequence of allcine phosphatase of 
Escherichia coil", pages 434-439 



Inventor: Pal, Rong-Chang 

1136 BIythe Street 

Foster City California 94404(US) 

Inventor: Rey, Michael Willard 

611 A Prospect Row 

San Mateo California 94403(US) 



0 Representative: Armitage, Ian Michael et al 
MEWBURN ELLIS & CO. 2/3 Cursitor Street 
London EC4A1BQ(GB) 



2 



EP 0 177 343 B1 



Description 

This application relates to methods for manufacturing proteins in bacteria. It particularly is concerned 
with isolating eukaryotic proteins from the periplasm of transformed bacteria while minimizing proteolytic 

5 degradation of the protein and contamination by nonperiplasmic proteins. This application also relates to the 
synthesis of mature eukaryotic proteins in bacterial hosts. It is directed to providing vectors that will express 
hybrid preproteins in high yields in host cells, cleave the signal sequence from the preprotein and secrete 
mature eukaryotic protein in the periplasmic space of the host cells. 

Literature that should be consulted in regard to this application is U.S. patent 4,375,514 and 4,411,994; 

10 U.K. patent application 2,091 ,268A (published 1982); I. Palva et al., "Gene" 22: 229-235 (1983); H. Inouye et 
al.. "J. Bact" 148(2): 434-439 (1983); K. Talmadge et al.. "P.N.A.S. USA" 77(7): 3988-3992 (1980); K. 
Talmadge et al., "P.N.A.S. USA" 77(6): 3369-3373 (1980); European Patent Application 114.695; R. Picken 
et at., "Infection and Immunity" 42(1): 269-275 (1983); T. Silhavy et al., "Microbiological Reviews" 47(3): 
313^344 (Sept. 1983); 3. KadonagTet al., "J. Biol. Chem." 259(4): 2149^2154 (Feb. 1984); Inter nationafPCT 

75 application WO 84/00774 (Mar. 1984); 0. Zemel-Dreasen et al.. "Gene" 27: 315-322 (1984); S. Michaelis et 
al., "J. Bact." 154(1): 366-374 (Apr. 1983); European Patent"ApplicationTl 4,695 (published Aug. 1, 1984); 
and G. Gray et al., "Biotechnology" pp 161-165 (Feb. 1984). 

Many naturally occurring secretory and membrane proteins are initially synthesized as nascent or 
intracellular preproteins. These are proteins in which a "signal" polypeptide is linked to the amino acid 

20 residue that will become the amino terminus of the mature protein upon secretion. The signal polypeptide is 
a peptide sequence which enables the mature protein to pass through the cellular membrane of the cell. 
The signal peptide is cleaved away, or "processed", in passing through the cellular membrane by a 
mechanism that is under study. If the processing occurs properly the mature protein will be free of any 
amino terminal extraneous signal amino acid residues and wit) have the proper amino terminal amino acid. 

25 Thus, if a heterologous gene which includes the DNA encoding a signal sequence is expressed by a host 
gram negative bacterial cell and the signal is then cleaved properly by the host, the mature protein without 
an appended methionine moiety is secreted into the periplasmic space of the host, i.e., the space between 
the inner, or cytoplasmic, membrane and the outer membrane of the host. Also known are host-vector 
systems in which the signal protein and at least an amino terminal portion of the mature protein ordinarily 

30 associated with the signal is expressed and processed while linked to a heterologous protein, thereby 
resulting in the secretion of a.fusion protein. 

Secretion of mature eukaryotic protein into the periplasm of gram negative bacteria such as E. coli has 
been an objective of the art for a number of years. Periplasmic secretion is a desirable objective because 
the product is thereby compartmentalized between the inner and outer cell membranes of the culture cells 

35 and not exposed to the rigors of the extracellular medium. Exposure to these rigors, e.g.. dilution, 
unfavorable salts or pH, oxidation, foaming, mechanical shearing and proteases, has handicapped the 
commercialization of nonperiplasmic secretion systems such as those using bacillus or yeast. Also, 
periplasmic compartmentalization simplifies recovery and purification of the desired mature eukaryotic 
protein. 

40 Heretofore the vector constructions employed for periplasmic secretion in gram negative bacteria have, 
to applicants' knowledge, all entailed the use either of complete eukaryotic signals or of partial hybrids. 
Constructions which encode two typical partial hybrids are schematically shown below, with the secretion 
cleavage sites generally encountered being designated with an arrow. 

46 



50 



55 



3 



EP0 177 343 B1 



70 



75 



Prokaryotic Signal 



Prokaryotic Signal 
Fragment 



Fragment of the 

Prokaryotic 
Mature Protein 



Vector- 



Mature Eukaryotic 
Protein 



Eukaryotic Signal 
or Fragment 



Mature Eukaryotic 
Protein 



-Vector- 



20 



25 



30 



Constructions like the partial bacterial preprotein-mature eukaryotic protein fusions shown in schennatic A 
result in the secretion of fusions of the prokaryotic and eukaryotic proteins. It is inconvenient and 
sometimes impossible to remove the extraneous amino terminal amino acids encoded by the prokaryotic 
DNA. On the other hand, constructions like those of schematic B, result in the secretion of mature protein 
but yields of secreted mature eukaryotic protein are less than desired. It would be useful to construct hybrid 
vectors as described below which could be expressed as direct hybrids of the prokaryotic signal and 
mature eukaryotic protein, processed by hosts and secreted into the periplasm. Such a vector Is shown 
below. 



Prokaryotic Signal 
Fragment 



Mature Eukaryotic 
Protein 



Vector— — — r-i 

35 J. Kadonaga et al., op cit, prepared a vector of the direct hybrid type In which the beta lactamase signal 
was linked directly lb DNA~for mature chicken triose phosphate isomerase. However, the isomerase was 
apparently neither secreted nor processed but resided as a hybrid protein both free in the cytoplasm and 
attached to the cytoplasmic side of the inner membrane. These authors concluded that the amino acid 
sequence of at least the early part of a mature secreted bacterial protein is critical to secretion across the 

40 Inner membrane of E. coli. 

The reasons for the relative difficulty previously encountered in obtaining the secretion of the product of 
direct hybrids are unknown. However, applicants speculate that while secretion and processing machinery 
are conserved In bacteria (so that eukaryotic preproteins have been processed In several cases), or the 
machinery is somewhat flexible (as shown In processing of schematic A-type fusions), a particularly difficult 

45 obstacle to secretion would appear to be posed by constructions in which such bacteria must process the 
expressed product at a completely artificial cleavage site, i.e., a site which is neither a prokaryotic nor 
eukaryotic site for about from 1 to 3 residues on either side of the point at which hydrolysis occurs. 
Contrary to this expectation, and the prior failures and skepticism of those skilled In the art, applicants have 
found that periplasmic bacteria indeed can process direct fusions of prokaryotic signals with mature 

50 eukaryotic proteins, and can do so with secreted mature protein yields higher than those obtained by 
applicants with eukaryotic preproteins. Particularly satisfactory results have been obtained in secreting 
mammalian growth hormones. 

Mammalian growth hormone is a normal product of the pituitary gland. Mammalian growth hormones 
are now known to exhibit a degree of species cross-specificity, a function of similar amino acid sequence 

55 and conformation. Human growth hormone (hGH) consists of 191 amino acids and has a molecular weight 
of about 21,500. HGH is in clinical use for the treatment of hypopituitary dwarfism. It also has been 
proposed to be effective In the treatment of burns, wound healing, dystrophy, bone knitting, diffuse gastric 
bleeding and pseudoarthrosis. 



4 



EP 0 177 343 B1 



Recently, hGH has been synthesized in recombinant host cells. See, for example, U.S. 4,342,832. HGH 
is not significantly degraded by bacterial cells and can be produced directly If the gene for its direct 
expression, including the appropriately placed start codon, is linked to a suitable promoter. Because 
prokaryotes frequently do not remove the amino-terminal methionine from the resulting protein, expression 

5 of heterologous hGH DNA under control of a bacterial promoter as shown in U.S. Patent 4,342,832 yields 
hGH having methionine as its first amino acid. The reason for this is that the DNA ATG codon (start codon) 
is ultimately expressed as methionine. Results to date, for example, with production of hGH in E. coll. have 
shown that the host cell has only a limited intracellular ability to cleave methionine from hGH "and only 
limited techniques presently exist to do so in vitro. 

70 E.P-0. Publication Number 127305 provides for the synthesis and secretion of mature hGH in 
prokaryotic hosts by transforming such hosts with prehGH, i.e., with hGH having its normal eukaryotic signal 
sequence. Host cells were able to express prehGH, to recognize the eukaryotic signal and to process the 
preprotein properly. Mature hGH was then recovered from the periplasm, but yields were not as high as 
desired. 

76 DNA sequences encoding a prokaryotic secreting signal sequence operably linked to the 5' end of DNA 
coding for eukaryotic proteins or for mature hGH are also described in the EP-A-55 942 and WO-A- 
84/03519 

One currently used technique for recovery of periplasmic protein is called spheroplasting (H. Neu et al., 
1964, "Biochem. Biophys. Res. Comm." 17: 215). This process entails the use of lysozyme to lyse^the 

20 bacterial wall, it is not attractive particularly for large scale recovery of therapeutic proteins because it 
entails the addition of another contaminant protein to the periplasmic extract and the spheroplasts are 
mechanically and osmotically fragile. Further, lysozyme is relatively costly. 

Another method is called osmotic shock (H. Neu et al., 1965, "J. Biol. Chem." 240(9): 3685-3692). This 
is disadvantageous principally because it requires two steps, first treatment of viable celts with a solution of 

26 high tonicity and second with a cold water wash of low tonicity to release the periplasmic proteins. 

These methods have been practiced on viable cells. The use of viable cells is undesirable because 
their proteolytic enzymes are fully active. When applicants attennpted to recover secreted hGH from a viable 
culture of E. coll, a proteolytic clip of unknown origin removed the amino terminal phenylalanine from about 
10 to 20 percent of the hGH. 

30 Accordingly, it is desirable to provide improved methods to recover periplasmic proteins. Such 
improved methods would minimize proteolytic degradation by proteases during recovery, and in general 
would be sufficiently delicate to minimize contamination of periplasmic protein by intracellular proteins. 
They also would permit the recovery of large proportions of periplasmic protein by manipulative steps more 
amenable to commercial, large scale use than currently available procedures, and would not entail the use 

35 of contaminating proteinaceous reagents. 

It is also desirable to express and secrete high periplasmic amounts of mature eukaryotic protein in 
bacterial hosts. 

It is further desirable to obtain periplasmic mature human growth hormone in elevated amounts. 
In a first aspect the invention provides a hybrid DNA sequence encoding a protein having at least the 
40 amino terminal sequence of mature hGH operably linked to a DNA sequence encoding an enterotoxin 
signal. 

DNA encoding E.coli enterotoxin signals are particularly useful in this regard. This signal DNA is 
characterized by not being linked (a) at its 3' end to the 5' end of DNA encoding mature enterotoxin or (b) 
at its 5* end to the 3* end of the enterotoxin promoter. It is conveniently employed as a cassette in the 
46 construction of enterotoxin signal-containing vectors. A preferred enterotoxin is STII. 

The STII Shine-Dalgarno (S.D.) sequence is a particularly powerful ribosome binding site which 
contributes to yield improvement. It ordinarily is linked to prokaryotic promoters such as the tryptophan (trp) 
or bacterial alkaline phosphatase (AP) promoters, and could be employed with any promoter system. 

In a second aspect the invention provides a replicable vector containing, in 5* to 3' order, a promoter, a 
50 Shine-Dalgarno sequence, a hybrid DNA sequence as defined above and a termination region comprising a 
stop codon; said promoter controlling transcription of the hybrid DNA sequence. 

In a third aspect the invention provides a method comprising; 

(a) constructing a vector comprising a DNA sequence encoding a protein having at least the amino 
terminal sequence of mature hGH operably ligated to a DNA sequence encoding an enterotoxin signal, 

55 the DNA being operably linked to the STII Shine-Dalgarno sequence; 

(b) transforming a host cell capable of processing the hybrid and secreting the mature eukaryotic protein 
with the vector; 

(c) culturing the transformed host cell; and 



5 



EP 0 177 343 B1 



(d) recovering the mature protein which comprises at least the amino terminal sequence of hGH from the 
periplasm of the cell. Desirably said step of recovering the mature protein from the periptasmic space 
comprises: 
(a) killing the cell; 
6 (b) freezing the cell; 

(c) thawing the cell; and 

(d) separating the periplasmic proteins, including the eukaryotic protein, from the remainder of the cells. 
In a fourth aspect the invention provides a prokaryotic cell culture comprising; 

(a) a mature eukaryotic protein and 
10 (b) a hybrid DNA encoding a direct fusion protein of an enterotoxin signal directly fused at its carboxyl 
terminus to the amino terminus of the mature eukaryotic protein; and wherein the mature protein Is 
located in the periplasm of the cell. 

In a fifth aspect the invention provides a prokaryotic cell culture comprising; 
(a) mature hGH and 

75 (b) a hybrid DNA encoding a direct fusion protein of an enterotoxin signal directly fused at its carboxyl 
terminus to the amino terminus of mature hGH; and wherein the mature protein Is located In the 
periplasm of the cell. Preferably said DNA sequence encodes the full sequence of mature hGH; and said 
recovery step (d) comprises: 

(e) killing the cell; 

20 (f) freezing the cell; and 

(g) separating periplasmic protein from the remainder of the cell. 

New prokaryotic cell cultures are produced upon transformation and culture of host cells using the 
above method. These cultures comprise (a) mature eukaryotic protein and (b) a direct hybrid fusion protein 
of the mature eukaryotic protein with a prokaryotic secretion signal sequence. Ordinarily, greater than about 
25 25 percent, generally up to about 90 percent, of the total weight of mature and fusion protein is mature 
protein located in the periplasm of the cell. 

The preferred method comprises obtaining viable or killed cells which have been transformed to secrete 
a heterologous or eukaryotic protein, causing the outer membrane of the cells to become permeable for 
passage of the protein out through the membrane as for example by freezing and thawing, and separating 
30 the periplasmic proteins, including secreted eukaryotic protein, from the remainder of the cells. It also Is 
advantageous to culture the cell under phosphate-limiting conditions to engender changes in the cell 
membranes which enhance recovery of the periplasmic protein. 

A novel killing method is provided wherein transformed cells are contacted with an alkanol and heated. 
These cells then are treated by a cold shock method comprising freezing and thawing which enables 
35 recovery of periplasmic proteins. 

These methods are employed to particular advantage in recovering hGH from gram negative organisms 
which express and process a direct hybrid fusion of the E. coli STII enterotoxin signal with mature hGH. 

Brief Description of the Drawings 

40 

Fig. 1 discloses the STII gene, including its translated and untranslated regions. The principal portion of 
its S.D. sequence is overlined at nucleotides 155-161. The imputed amino acid sequence for the STII signal 
is located at residues -23 to -1 and for the mature STII enterotoxin at residues 1-48. Fig. 1 also discloses 
the processing site for STII (designated "cleavage site") and various restriction enzyme sites. The asterisk 
45 designates the likely mRNA synthesis initiation site assuming that the STII promoter includes the overlined 
structures at position 84 - 89 and 108 - 114. 

Figs. 2a - 2d disclose the construction of a vector (ptrp-STll-hGH) encoding a secretable STIl-hGH 
fusion protein under the control of the trp promoter and containing an STII S.D. sequence. 

Fig. 3 is a detail of the plasmid tr-STII-hGH. 
60 Fig. 3a is the nucleotide sequence of the trp promoter region, STII signal and the hGH gene in ptrp- 
STll-hGH. 

Figs. 4a-4c disclose the construction of the vectors pAP-1 and pAP-STII-hGH encoding secretable AP- 
, hGH and STIl-hGH fusion proteins under the control of the AP promoter, the vector encoding the latter 
containing an STII S.D. sequence. 
55 Fig. 5 is the nucleotide sequence of the AP promoter region, STII signal and the hGH gene in pAP-STII- 
hGH. 

Fig. 6 is the nucleotide sequence of the AP promoter region, the AP signal and the hGH gene in pAP-1. 



6 



EP 0 177 343 B1 



Detailed Description 

A heterologous protein is a protein not ordinarily secreted by the bacterial cell. While such a protein 
may be an ordinarily intracellular protein of the transformed cell that has been engineered for secretion, or it 
5 may be a protein encoded by DNA from another microbe, ordinarily it is a eukaryotic protein, a fragment 
thereof or a fusion thereof with a prokaryotic protein or fragment. Preferably the periplasmic protein Is a 
mature eukaryotic protein such as a hormone, e.g., hGH, an interferon or a lymphokine. 

The cells to be treated herein may be obtained from cultures of transformed bacteria grown in 
conventional media or media tailored to the vectors or mutant host transformants used to secrete the 
70 heterologous protein. 

Applicants have demonstrated the secretion of direct hybrid fusions of a prokaryotic signal and desired 
eukaryotic protein, notwithstanding the complexity of the host vector systems at both the level of preprotein 
synthesis and secretion. Plasmids were constructed in which the trp or AP promoters were employed with 
various directly linked prokaryotic signal and eukaryotic protein sequences. Suitable hosts were transformed 
75 with each of these plasmids and the amount and distribution of product between the periplasm and 
cytoplasm determined. These experiments demonstrated that mature eukaryotic proteins are secreted and 
correctly processed into the periplasmic space of organisms from direct hybrid fusion proteins. The 
following table shows the results of successful experiments. The results obtained by using the normal hGH 
eukaryotic signal are shown for comparison, 

20 



25 



30 



35 



40 



45 



50 



55 



7 



EP 0 177 343 B1 



Table 1 

Effect of Promoters and Signal Peptides on 
Expression and Secretion of Eukaryotic Proteins 



Promoter Gene Locat ions/Levels3 Media 

Signal Host Processing^ 



trp ST 11 



AP 



AP 



trp 



STII 



AP 



hGH 



trp STII 



trp STII 



hGH 



hGH 



hGH 



hGH 



294 or 
W31108 



W3110 



294 or 
W3110 



hLIF-AS 294 
mIgG-K? 294 



90 percent 
perlplasmic; 
1 gram 

50 percent 
perlplasmic; 
0.5 gram 

90 percent 
perlplasmic; 
0.1 gram 

90 percent 
perlplasmic; 
50 mg 

50 percent 
perlplasmic; 
1.9 mg 

60 percent 
perlplasmic; NO 



correct 



correct 



correct 



correct 



correct 



ND5 



defined or LB 



def ined-pil 



LB-pi 



def ined/LB 



LB 



LB 



inorganic phosphate depleted medium 
E. coll ATCC 31446 

Variation is typically encountered among experiment!. Relevant 
parameters include culture density, the time at which secreted 
protein is recovered, and other variables. The total amount of 
mature and fused eukaryotic protein, as well as the periplasmic 
percentage, must be considered approximate. The levels are 
reported as grams/SO equivalent culture OD units at 550 
nm/liter. Cultures were grown in 10 or ,20 ml fermentations in 
shake flasks. 

Correct processing means that the secreted protein exhibited 
the same amino terminus as is found when the protein is 



8 



EP 0 177 343 B1 



isolated from natural sources. 

ND means not done. 

human leukocyte interferon - A 

The light chain of a murine monoclonal anti-CEA immunoglobulin, 
E. coli ATCC 27325 



Applicants' initial attempts to secrete some mature eukaryotic proteins using prokaryotic signals in the 
above structural format did not result in synthesis of protein in some cases, and in others expression was 
not accompanied by secretion. Cultures transformed with vectors for the murine immunoglobulin heavy 
chain (using the STII signal), human tissue plasminogen activator (using the AP promoter and signal) or a 

76 bovine prorennin (using the trp promoter and the STI! signal in E. coli hosts), or hGH (using the 
Pseudomonas aeruginosa enterotoxin A signal) synthesized very low or undetectable quantities of either the 
preprotein or secreted mature protein. Other transformed cultures failed to process STII signal fusions with 
bovine gamma interferon, prorelaxin, interleukin-2 or bovine prorennin. These results were obtained in 
limited and preliminary experiments. 

20 It is clear from this work that direct hybrid fusions of prokaryotic signals with mature eukaryotic proteins 
are recognized by bacterial cells, i.e., processed and transported into the periplasm. Given that knowledge, 
the skilled artisan must nonetheless exert diligence in identifying functional constructions. 

This effort should be directed first at screening vectors encoding direct hybrid fusions with signals 
obtained from a variety of periplasmic bacterial proteins, e.g. enzymes or enterotoxins. If the secretion of 

25 mature eukaryotic protein is not obtained upon the transformation and culture of E. coli with such 
constructions then other genera of gram negative bacteria should be screened for the abllitylo secrete the 
mature protein. Lastly, the signal peptide may be mutated in order to enhance or modify its processing 
characteristics. 

The method herein is facilitated by the ability of gram negative bacteria to recognize direct fusions with 
30 the STII enterotoxin signal peptide. Furthermore, it is based on the additional discovery that elevated yields 
are obtained by use of the STII S.D. sequence. 

Partial amino acid sequences for hGH and STII enterotoxin preproteins are shown below with the 
starting amino acid for each mature protein being underlined. 

36 hGH ... leu gin glu gly ser ala phe pro ala met ser leu... 
STII ... ala thr asn ala tyr ala ser thr gin ser asn lys... 

As can be seen, virtually no homology whatever exists between these two sequences in the vicinity of their 
signal cleavage sites. Thus it was surprising that the host cells were able to recognize the STII-hGH hybrid 

40 junction and process the STII-hGH preprotein correctly and secrete hGH. 

The prokaryotic signal sequence to be used herein is the signal sequence from any bacterial secreted 
or cell membrane enterotoxin, or mutation thereof. The preferred embodiments are found among the heat 
stable (ST) and heat labile (LT) enterotoxins of E. coli. Of the ST enterotoxins, STII is most preferred. 

According to Picken et al., op cit, the STII signal polypeptide was either the amino acid sequence NH2- 

46 met tys lys asn ile ala phe leu leu ala ser met phe val phe ser ile ala thr asn ala, or this sequence with an 
additional carboxyl-terminal tyr ala. In fact, E. coli cleaves the signal after tyr ala. Thus, the STII signal DNA 
which is used in the vectors further described hirein will encode the tyr ala alternative. 

The prokaryotic signal may be mutated in order to increase the proportion of eukaryotic protein that is 
secreted. Generally, mutations in codons encoding amino acid positions outside of the hydrophobic ^ore of 

50 the signal, e.g., residues -1, -2 or -3, are more likely to exert a significant effect on signal cleavage. The 
mutated DNA will express an amino acid at the site of mutation which is different from the wild type signal. 
Most conveniently a plurality of codons are substituted at a given position, each of which encodes a 
different amino acid. This Is accomplished by methods known in the art. For example see S. Michaelis, op. 
cit., as applied to the alkaline phosphatase signal. Each individual construction then is ligated to DNA 

56 encoding the mature human protein in an expression vector, the vector used to transform hosts, the 
transformants cultured and the secretion level determined for each mutant as is more fully described below. 
Transformants that secrete optimal levels of desired protein are identified and the responsible constructions 
selected. With respect to the STII signal, it is expected that amino acid substitutions in the hydrophobic 



9 



EP 0 177 343 B1 



region (residues -5 to -17 in Fig. 1) will not adversely affect the signal efficacy so long as the substituted 
amino acids are uncharged and, preferably, hydrophobic. Also, deletions or insertions of one or two like 
residues in this region are acceptable. The most sensitive region of the leader is residues -1 and -21 to -22 
(Fig. 1). Mutations in these residues, including insertions or deletions generally are deleterious, but 

6 occasionally beneficial effects on yield or secretion are obtained. Mutations that are so extensive as to 
convert the expressed signal to the eukaryotic signal ordinarily associated with the desired protein or other 
eukaryotic proteins are not prokaryotic signal mutations as defined herein. 

Mutagenic optimization is particularly desirable upon switching from one host to another while using 
vectors containing substantially the same signal. The reason for this Is that allelic variants In the bacterial 

70 enzyme system responsible for cleaving the signal-eukaryotic protein fusion and transporting the mature 
protein may more readily recognize a suitably modified prokaryotic signal. 

In accordance with this Invention any eukaryotic protein, mutant or derivative may be secreted if proper 
gram negative hosts and signals are selected, provided of course that the protein per se is capable of being 
expressed in gram negative hosts. Such eukaryotic proteins Include lymphokin'iil Immunoglobulins and 

75 hormones. The eukaryotic proteins used herein ordinarily are mammalian proteins such as those of bovine, 
porcine and human origin. Particularly advantageous results are obtained with growth hormones such as 
human growth hormone. 

Vectors for transforming hosts to express direct hybrid fusion proteins are made in conventional fashion 
by methods generally known to those skilled in the art. In such vectors. DNA encoding the prokaryotic 

20 signal Is directly linked to DNA encoding the desired protein. This means that the signal DNA encoding the 
amino acid immediately upstream from the normal bacterial cleavage site is linked directly to the DNA 
encoding the first amino acid of the desired mature eukaryotic protein, without any intervening residual 
sequences from the mature prokaryotic protein or the normal eukaryotic pre sequence. Such direct linkage 
is accomplished by known methods such as the Ml 3 deletional mutagensis procedure described in the 

P5 examples. Alternately, one may chemically synthesize DNA encoding the signal and cleavage region. This 
DNA is blunt end llgated, or ligated through a convenient restriction enzyme site, to DNA encoding the 
remainder of the eukaryotic protein. 

DNA encoding STII signal Is directly linked in either fashion to DNA encoding the desired mature 
eukaryotic protein. When the eukaryotic protein is a growth hormone, the signal will be linked to DNA 

30 having as its two first 5' codons. codons which encode at least the first two amino-terminal amino acids of 
hGH, i.e.. NH2-phe pro, and preferably encode the first 15 amino-termlnal amino acids of hGH. This 
sequence ordinarily is part of a DNA sequence encoding growth hormone such as hGH, bovine growth 
hormone (having the amino-terminal sequence phe pro ala met ser leu) or porcine growth hormone (having 
the amino-terminal sequence phe pro ala met pro leu) and the allelic variants thereof. 

35 Suitable vectors for use herein are constructed by operably ligating the direct hybrid fusion DNA to 
replication and translation effecting sequences. Sequences which effect translation of the mRNA include an 
S.D. sequence present upstream from the prokaryotic signal. Preferably, the S.D. sequence used herein Is 
that of STII, and it is spaced from the prokaryotic signal start codon by the natural intervening sequence 
(TTTT) found in the STII gene. This construction is obtained most easily by isolating the STII signal 

40 complete with its S.D. sequence and normal Intervening sequence from a source of the STII gene as is 
further described below. However, because this portion of the STII gene is only 80 bp long it also is 
practical to simply synthesize the required sequence by known chemical methods. This Is especially 
preferred if extensive mutagenesis of the signal is contemplated. The construction shown in Fig. 3a contains 
two S.D. sequences, one upstream S.D. sequence donated by the trp promoter and then the STII S.D. 

45 sequence in its normal relationship with the STII signal DNA, We believe that the upstream, non-STII S.D. 
sequence is not required. Other prokaryotic signal sequences also may be synthesized by chemical 
methods as they tend to be less than 100 bp In length. 

Transcription of the foregoing S.D.-signal-protein sequence is under the control of a promoter. The 
promoter is preferably a prokaryotic promoter other than the promoter ordinarily associated with the 

50 selected prokaryotic signal. The preferred embodiment is the AP promoter, although others such as the tac, 
trp or lactose (D. Goeddel et al., "Nature" 281: 544 [1979]) promoters are satisfactory. While these are the 
most commonly used, other microbial promoters have been discovered and utilized, and details concerning 
their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with 
plasmid vectors (Siebenlist et al., 1980, "Cell" 20: 269). The promoter does not appear to affect the 

55 proportion of eukaryotic protein that is secretedT However, it is desirable to screen combinations of 
promoters and signal sequences for optimal expression since both elements interact in affecting expression 
levels. For example, compare the results in Table 1 when the STII signal Is substituted for the AP signal in 
combination with the AP promoter. 



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The promoter-S.D.-signal sequences are present in vectors containing replicon sequences compatible 
with the host cells. The vectors are generally plasmids rather than phage. They contain a replication site as 
well as phenotypic marking sequences to facilitate identification of transformed cells. For example, E. coli is 
typically transformed with a derivative of pBR322, (Bolivar et al., "Gene" 2: 95 [1977]), a plasmid containing 

5 genes for ampicillin and tetracycline resistance. 

When a DNA sequence is "operably ligated or linked" to another it means that the DNA sequence in 
question exerts an influence on other DNA sequences. This generally means that the first DNA controls 
either the transcription or ultimately the translation of the DNA to which it is operably ligated, or affects the 
processing of the translated protein. For example, a promoter is operably ligated to DNA encoding a 

70 preprotein when it affects the rate of translation of the preprotein mRNA. A signal polypeptide is operably 
ligated to DNA encoding a desired protein when it is placed in reading frame with and ligated directly to the 
DNA encoding the protein. The term "ligated" in reference to a composition should not be inferred to mean 
that the composition is only defined in terms of its possible manufacture by ligation. On the contrary, 
operably lighted elements in a plasmid can be synthesized chemically as a unitary entity. 

75 The host cells that are transformed with the foregoing vectors are bacteria (a) having a periplasmic 
space between two cell membranes, (b) in which the vector replicates and (c) in which the preprotein is 
both expressed and processed. The hosts are generally gram negative organisms, particularly Enterobac- 
teriaceae or Pseudomonas and mutants thereof. Host vector systems (hosts transformed with vectors) are 
preferred in which the promoter controlling expression of the hybrid fusion is constitutively activated or 

20 derepressed. A constitutive mutant is one that is capable of secreting a given protein, which here is the 
promoted direct hybrid fusion and, in some cases, the normally promoted protein, without induction or other 
changes in culture conditions calculated to derepress or activate the promoter. Either the host cell or the 
promoter, or both, may be mutated in order for the host-vector system to be constitutively promoted. The 
promoter used in the vector may be mutated so that it is no longer capable of being repressed by a 

26 repressor protein. Such mutations are known. Alternatively, the host cells are mutated to become constitu- 
tive for proteins under the control of a wild-type or unmutated promoter. Such hosts are conveniently 
prepared by transduction from publically available strains containing the mutant alleles. Preferably, host 
cells transformed with AP jDromoter-bearing vectors carry a phoT or phoR mutant allele. The former is a 
disabling mutant believed to be in the gene encoding a phosphate ion transport protein. The phoR mutant is 

30 a disabling mutant in the gene encoding a repressor protein. These mutant alleles are widely known and 
available in the art and may be transfered Into hosts by known techniques. Surprisingly, the constitutive 
hosts processed a greater percentage of the expressed product than the wild type hosts which were 
induced by phosphate depletion. 

The host cells may, but need not constitutively express the host protein normally promoted by the 

36 promoter used to control transcription of DNA for the direct hybrid fusion. For example, either an E. coli 
phoA deletion mutant, which fails to express active AP, or an AP-synthesizing cell may be used as host; the 
active secretion of AP by the cells does not necessarily interfere with secretion of the eukaryotic protein. 
Ordinarily, the host bacterium will be the same species from which the signal was obtained. 

While any culture medium ordinarily used for the host cells is satisfactory for the transformants, the 

40 composition of host culture medium exerts a strong effect on the secretion of eukaryotic protein by wild 
type hosts (rather than strain W3110 phoA, phoT or phoA, phoR). Media containing yeast extract results in 
improved secretion when compared to tryptone (a tryptic digest of casein) or a synthetic mixture of 19 
amino acids. It appears that one or more components of yeast extract helps to activate secretion. 

In the preferred embodiment the cells are to be grown under phosphate limiting conditions prior to 

45 harvest of the culture. This is the case whether or not the organism is transformed with a vector encoding a 
eukaryotic protein under the control of a promoter for a phosphate processing protein, or whether the cell is 
defective in transporting or catabolizing phosphate nutrients. Surprisingly, the use of a phosphate-limiting 
culture medium prior to cell harvest during the late stages of fermentation greatly improves the mechanical 
characteristics of transformed gram negative organisms. These characteristics enhance the efficacy of the 

50 cold shock extraction method described infra. The medium is made phosphate limiting, for example, by 
either precipitating the phosphate from the medium, by introducing phosphate to the culture at a limiting 
feed rate, or by providing an Initial supply of phosphate that will be inadequate to support optimal growth of 
the culture beyond a certain predetermined density. The initial supply will be sufficient to support optimal 
cell growth during early stages of the fermentation, for example log phase growth or to an OD550 of about 

56 45. The amount of phosphate will be tailored to the other nutrients and to the strain of organism used in the 
culture. Ordinarily, about 2.5 g/l of potassium/sodium phosphate salts are employed in media for use with E. 
coli W3110 (ATCC 27325), but larger amounts (about 4.0 g/l) are used with cells having phosphate 
metabolism defects such as the phoT mutant. This is in contrast with nonlimiting quantities of phosphate 

11 



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(about 9.0 g/1). The amount of phosphate ion in the limiting culture medium at the point the culture becomes 
limiting is less than about 1 mM. Typically the cells are cultured under these limiting conditions for at least 
about 1 hour prior to harvest. 

Preferably the cells are permitted to accumulate periplasmio heterologous protein to a maximum level. 

5 usually during a growth stage following logarithmic growth, prior to initiation of the treatment provided 
herein. The cells should be killed as soon as this point is reached or shortly thereafter. The term "killed" 
means that the cells at least are unable to replicate. It is likely, however, that treatment with alkanol and 
heat as is further described infra interdicts or destroys metabolic functions not directly associated with 
replication. However, other than elimination of a deleterious proteolytic activity of E. coli towards periplasmic 

70 hGH the impacted metabolic functions remain unknown. The killing procedure shoTjId not rupture, lyse or 
weaken the inner cell membrane of the host organism. 

Cells that have reached the desired point of growth are preferably killed by immediately contacting 
them with an alkanol and heating. The alkanol should not be an alkanol which is lytic for cell membrines, 
e.g. 1-octanol. Generally, suitable alkanols include lower (C2 to C4) monohydroxy alkanols such as 1-butanol 

75 or ethanol, preferably 1-butanol. The alcohol should be contacted with the cells by adding the alkanol to the 
fermentation culture while continuously mixing. 

The amount of alcohol which is added will be such as to bring the culture to an alkanol concentration of 
about from 0.5 percent to 10 percent vol/vol, and preferably about to 1.5 percent vol/vol for 1-butanol. 
Butanol is preferred because as little as about 0.5 percent can be used; larger proportions are needed for 

20 equivalent inactivation when using a propanol or ethanol. 

Contemporaneous with or after the addition of alkanol to the cell culture, the temperature of the culture 
is increased to and held at a level sufficient in conjunction with alkanol to kill the cells. Ordinarily this will be 
about from 0.5 to 20 minutes at a temperature of about from 55* C to 35' C, the higher temperatures being 
employed for the lesser periods. The alkanol enables the heating to be conducted at a lower temperature 

25 than would otherwise be required, an advantage in preserving the activity of proteins to be recovered. 

Cell killing is not critical. Since it is useful in preventing or retarding product degradation it may be 
dispensed with if the cells can be processed quickly and regulatory requirements for handling recombinant 
cells can be otherwise complied with. However, we have found that killing the cells approximately doubles 
the product protein recovery without reducing the purity of the product protein in the recovered super- 

30 natants. 

After the cells are cultured and/or killed, periplasmic protein is recovered. This generally entails forming 
a paste of the cells, freezing the cells, thawing the cells, suspending them in buffer, and separating the 
periplasmic proteins from cell debris, e.g. by low speed centrifugation or filtration. The periplasmic proteins, 
including secreted eukaryotic proteins, are located in the supernatant. Eukaryotic periplasmic proteins may 

35 be obtained in higher specific activity, i.e. purity, than is attained with osmotic shock methods, and 
treatment with a hypertonic agent such as 20 percent sucrose is not required, but it should be understood 
that any method for causing the outer membrane of the cell to become permeable to the periplasmic 
protein can be used with killed cells. 

The cell paste to be frozen is produced by centrifuging or filtering the cell culture and recovering the 

40 cell mass. This paste typically contains residual quantities of the fermented culture medium, e.g. LB (Luria 
Broth) medium; it is unnecessary to wash the cells with a freezing menstruum prior to the subsequent 
steps. It is not critical that a cell paste be formed as the cold shock extraction method herein is largely 
independent of cell density. However, the economics of freezing and thawing large volumes of material are 
such that small paste volumes are preferred. 

45 The cell freezing should be as soon as possible. Generally the paste at room temperature is placed in a 
freezer at -20' C until frozen, and then stored at -80' C until further processing is required. 

The frozen paste is thawed and diluted into several volumes of water or aqueous buffer, preferably 
about 3 or greater volumes of 10 mM tris-HCI buffer at pH8. This buffer is hypotonic to the intracellular 
contents of the host organisms. The following steps are performed at about 4*C. The dilutions generally 

60 should be about 3 volumes of buffer, although the identity, concentration, and pH of buffer will vary 
depending upon the periplasmic protein to be recovered. 

The cells are thoroughly suspended in the buffer. This step is facilitated by suspending the cells in the 
buffer by use of a homogenizer set at a speed at which no substantial lysis of the celts occurs. 

The suspended cells are gently stirred for about from 10 to 60 minutes, ordinarily about 30 minutes. 

55 Then the cell debris and cells (containing cytoplasmic proteins) are removed by centrifugation, typically 
12,000 X g for 30 minutes, or by filtration. The supernatant contains the periplasmic proteins, solubilized 
outer membrane proteins, residual culture medium and extracellular proteins, and a small proportion of 
soluble intracellular proteins. The desired eukaryotic protein may be purified from the supernatant in accord 



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with further procedures as desired. 

The freeze-thaw extraction method described above offers considerable advantages in terms of yield 
and, surprisingly, purity of the desired perlplasmic protein when compared to extraction of either fresh killed 
or unkilled transformants with buffer alone. Approximately 5 to 26 times as much hGH is recovered from 
5 frozen unkilled cells than from unfrozen, unkilled buffer-extracted cells. The supernatants from frozen killed 
cells were about 19 percent hGH by weight of total protein, but from fresh killed cells only about 16 percent 
hGH. 

The foregoing method is applied in the following Examples to the recovery of hGH from E. coli 
transformed with a vector which expresses mature hGH as a direct hybrid fusion with the E. coli heat~stabie 

70 enterotoxin STII signal peptide. However, it will be appreciated that the recovery method herein Is 
applicable to the separation of periplasmic proteins from transformed bacterial cells in general, e.g. those 
disclosed in U.S. patents 4,411,994 and 4,375,514; K. Talmadge et al., op cit (both citations); 0. Zemel- 
Dreasen et al., op cit and G. Gray et al., op cit. 

In order to simplify the Examples certain frequently occurring and well-known methods employed in 

76 recombinant constructions will be referenced by shorthand phrases or designations. 

Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers. 
The starting plasmids or sources of DNA herein are commercially available, are publically available on an 
unrestricted basis, or can be constructed from available plasmids or polynucleotides in accord with 
published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the 

20 ordinary artisan since the plasmids generally only function as replication vehicles for the preprotein and its 
control sequences, or for elements thereof in intermediate constructions. 

"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain 
locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific 
is called a restriction site. "Partial" digestion refers to incomplete digestion by a restriction enzyme, i.e., 

25 conditions are chosen that result in cleavage of some but not all of the sites for a given restriction 
endonuclease in a DNA substrate. 

The various restriction enzymes used herein are commercially available and their reaction conditions, 
cof actors and other requirements as established by the enzyme suppliers were used. Restriction enzymes 
commonly are designated by abbreviations composed of a capital tetter followed by other letters and then, 

30 generally, a number representing the microorganism from which each restriction enzyme originally was 
obtained. In general, about 1 ug of plasm id or DNA fragment is used with about 1 unit of enzyme in about 
20 ul of buffer solution. Appropriate buffers and substrate amounts for particular restriction enzymes are 
specified by the manufacturer. Incubation times of about 1 hour at 37 are ordinarily used, but may vary 
in accordance with the supplier's instructions. After incubation, protein is removed by extraction with phenol 

35 and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with 
ethanol. Digestion with a restriction enzyme infrequently is followed with bacterial alkaline phosphatase 
hydrolysis of the terminal 5' phosphates to prevent the two restriction cleaved ends of a DNA fragment from 
"circularizing" or forming a closed loop upon ligation (described below) that would impede insertion of 
another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed 

40 by 5' terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional (T. 
Maniatis et al., 1982, Molecular Cloning pp. 133-134). 

"Recovery" or "isolation" Df a given fragment of DNA from a restriction digest means separation of the 
digest on polyacrylamide gel electrophoresis, identification of the fragment of interest by comparison of its 
mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section 

45 containing the desired fragment, and separation of the DNA from the gel, generally by electroelutlon. This 
procedure is known generally. For example, see A. Lawn et al. 1981, "Nucleic Acids Res." 9: 6103-6114. 
and D: Goeddel et al.. 1980, "Nucleic Acids Res." 8: 4057. 

"Southern Analysis" is a method by which the presence of DNA sequences in a digest or DNA- 
containing composition is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment. 

50 For the purposes herein, unless otherwise provided. Southern analysis shall mean separation of digests on 
1 percent agarose, denaturation and transfer to nitrocellulose by the method of E. Southern, 1975, "J. Mol. 
Biol." 98: 503-517. and hybridization as described by T Maniatis et al., 1978 "Cell" 15: 687-701. 

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an 
extrachromosomal element or chromosomal integrant. Unless otherwise provided, the method used herein 

56 for transformation of E. coli is the CaCh method of Mandel et al., 1970, "J. Mol. Biol." 53: 154. 

"Ligation" refers lb "the process of forming phosphodieste7 bonds between two double stranded nucleic 
acid fragments (T. Maniatis et al.. Id., p. 146). Unless otherwise provided, ligation may be accomplished 
using known buffers and conditi'ons with 10 units of T4 DNA ligase ("ligase") per 0.5 ug of approximately 



13 



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equimotar amounts of the DNA fragments to be ligated. 

"Preparation" of DNA from transformants means isolating plasmid DNA from microbial culture. Unless 
otherwise provided, the alkaline/SDS method of Maniatis et al., Id. p. 90., may be used. 

"Oligonucleotides" are short length single or double~stranded polydeoxynucleotides which are cheml- 
5 cally synthesized by known methods and then purified on polyacrylamide gels. 

All literature citations are expressly incorporated by reference. 

Example 1 

70 ^ Construction of a Plasmid Encoding for the E, coli Heat-Stable Enterotoxin (STII) Gene Signal Peptide 
Sequence. 



The following construction is illustrated In Fig. 2a. The plasmid p,WM501 (PIcken et al, op cit) contains 
the heat-stable enterotoxin (STII) gene. A portion of the DNA which encodes the STII~gene~was~recovered 

76 from pWM5p1 2 (stippled region of Fig. 2a) using the following steps. pWMSOl was digested with Rsal and 
the 550 bp DNA fragment 2 was isolated. This gene fragment was ligated to the phage M13mp8 (J. 
Messing et al. in the Third Cleveland Symposium on Macromolecules: Recombinant DNA , Ed. A. Walton, 
Elsevier, Amsterdam (1981) pp 143-153) that had been previously digested with Smal. The ligated DNA was 
used to transform E. coli JM101, a commercially available strain for use with the Ml 3 phage. Clear plaques 

20 were recovered. The "double stranded M13mp8 STII Rsa derivative 3 was isolated from an E. coli JM101 
infected with this phage using standard procedures (J. Messing et al. op cit). By the use of~the~M13mp8 
subcloning procedure just described the approximately 550 base paiFfragnnent 2 containing the STII leader 
gene is now bounded by a series of different restriction endonuclease sites pTovided by the phage. The 
M13mp8 STII Rsa derivative 3 then was digested with EcoRI and Pst I and a DNA fragment 4 slightly larger 

26 than fragment 2 was isolated." . 

EcoRI-PstI fragment 4 was subcloned into pBR322. This was accomplished by digesting pBR322 with 
EcoRI and PstI and isolating the vector 5. The isolated vector 5 was ligated to the EcoRI-PstI DNA fragment 
4. This DNA mixture was used to transform E. coli 294 and tetracycline resistant colonies selected. A 
plasmid 6 was isolated from a resistant E. coli colony and designated pSTII partial. 

30 

Example 2 

Construction of a Plasmid encoding the STII Signal Peptide Under the Control of the Trp Promoter. 

35 This construction method is shown in Fig. 2b. pSTII partial from Example 1 was digested with Mnll and 
BamHI and a 180 bp fragment 7 containing the STII S.D. sequence, the STII signal sequence, and the first 
30 codons of the mature STII gene was isolated. DNA fragment 7 was ligated to a plasmid containing the 
trp promoter. One such plasmid pHGH207-1, 8, has been described previously (H. de Boer et al., 1982, in: 
Promoters : Structure and Function , Eds. R. Rodreguez et al. Chamberlin, Praeger Pub., New" York, NY, pp 

40 462-481). A derivative of this plasmid, pHGH207-1*, wheTeirTthe EcoRI site 5' to the trp promoter had been 
converted to EcoRP by filling in with DNA polymerase I (DNA pol I) and joining the blunt ends by ligation (S. 
Cabilly et a!., 1984, "Proc. Natl. Acad. Sci. USA" 81: 3273-3277) was used in this example. The trp 
promoter-containing plasmid was digested with Xbal and treated with DNA pol I and all four dNTPs to fill in 
the protruding sequence. The DNA preparation was then digested with BamHI and the vector containing 

45 fragment 9 isolated. Vector fragment 9 then was ligated to the 180 bp STII signal-containing DNA fragment 
7 isolated above. The ligation mixture was used to transform E. coli 294 to ampicillln resistance. A plasmid 
designated STII leader 10 was isolated from an ampicillin resistant colony. This plasmid contains the STII 
signal sequence and a portion of the gene encoding mature STII under the control of the trp promoter. In 
the following example, the DNA sequence encoding mature hGH was operably ligated downstream from the 

50 trp - STII signal sequence. 

Example 3 

Construction of an Expression and Secretion Plasmid for hGH 

65 

Refer to Figs. 2c-2d for a schematic display of this method. STII leader 10 was digested with Bglll then 
treated with DNA pol I and all four NTP's to fill in the protruding end, and then digested with BamHI. The 
vector-containing fragment 11^ was isolated. The plasmid pHGH207-1 from Example 2 was digested with 



14 



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EcoRI, treated with DNA pol I and all four NTP's to fill in the protruding end, and then digested with BamHI. 
The hGH gene-containing fragment 12 of about 920 bp was isolated from the BamHI digestion. These two 
fragments were ligated and the DNA" mixture used to transform E. coli 294 to tetracycline resistance. A 
plasmid designated ptrpSTII-HGH-fusion 13 was isolated from the~re¥istant E, coli colonies. This plasmid 

5 still contains extraneous nucleotides encoding a portion of the STII mature protein between the STII leader 
peptide sequence and the hGH structural gene. These nucleotides were deleted using the Ml 3 site specific 
mutagenesis procedure (J.P. Adelman et al, 1983, "DNA" 2: 183-193). 

The gene from ptrpSTII HGH fusion 13 was incorporated into the single stranded phage M13mp10 (J. 
Messing et al., op cit. and J. Adelman et al. op cit.). Ml 3 mutagenesis phage and E. coli strains are 

70 commercially available. Mutagenesis was accom^ished first by digesting plasmid 13 with Xbal and BamHI 
and then recovering fragment 14. M13mp10 was digested with Xbal and BamHI iSd the phage fragment 
(not shown) was Isolated. Fragment 14 was ligated into the phage fragment and the ligation mixtures used 
to transform JM101. The transformed culture was plated and incubated. Phage having the fragment 14 
insert were identified as clear rather than blue plaques in the E. coli chromogenic indicator lawrT 

75 Corresponding phage were grown on E. coli JM101, and the culture centrifuged. Single stranded phage 15 
are present in the supernatant. Single'strahded phage 15 DNA was prepared, annealled to the synthetic 
oligonucleotide primer 5*pCAAATGCCTATGCATTCCCAACTATACC-OH3', primer extended with DNA pol I 
and the four NTPs to obtain double stranded DNA (one of which strands contained the deletion), treated 
with T4 ligase, extracted and used to transform E. coli JM101 (See J. Adelman et al., op cit). Note that the 

20 first 14 nucleotides of the primer are the coding sequence for the 3' end of the STlfsiglnairwhile the last 14 
nucleotides are the coding sequence for the 5* end of the mature hGH gene. Double stranded phage were 
obtained from the cellular contents of the transformed JM101, transfected into plated E. coli JM101, transfer 
filter impressions taken of the plates and double stranded phage containing the deletforTwere identified on 
the filters by Southern analysis with a 5*-32P-labelled obigonucleotide having the DNA sequence of the 

25 primer. Double stranded DNA 17 was prepared from the E. coli infected with the M13mp10 containing the 
gene deletion. This DNA was digested with Xbal and BarnHf^d the DNA fragment 17 isolated. This was 
ligated in the presence of fragment 18 from similarly digested and isolated pHGH207^1. The ligation was 
used to transform E. coli 294 to tetracycline resistance. Plasmid ptrp-STII-HGH 19 was recovered and its 
nucleotide sequence determined. A detailed restriction map of this plasmid is shown in figure 3. The DNA 

30 sequence of this plasmid in the vicinity of the hGH gene is shown in Fig 3a. 

Example 4 

Expression and Secretion of hGH 

35 

HGH was synthesized in shake culture using plasmid 19 from Example 3. E. coli 294 was transformed 
with plasmid 19 and innocuiated into 10-20 ml of LB medium with 5 ug/ml tetracycline in a 50 or 125 ml 
shake flask, fhe flask was cultured for 12-24 hours at 37 °C without the addition of any further medium, 
after which the cells were recovered by centrifuging. Total cellular hGH was assayed by radioimmunoassay 
40 of sonicated cells. The secreted hGH was recovered through osmotic shock and determined to be mature 
hGH by SDS-PAGE and N-terminal amino acid terminal sequencing. Amounts recovered were about ten 
times that which is expressed when using the human hGH signal under control of the trp promoter. 

Example 5 

45 

Construction of Plasmid pAP-1 Designed to Express and Secrete Human Growth Hormone (hGH) Under the 
Control of the AP Promoter and Signal Sequence. 



This construction is shown in Figs. 4a-4c. A DNA fragment containing a portion of the AP gene was 
60 isolated from the plasmid pHI-1 20 [Inouye, H., et a!., J. Bacteriol. 146: 668-675 ^1981)]. This was done 
using a series of steps to introduce an EcoRI site 5' to the AP gene and pronrioter sequence. The plasmid 
20 was digested with Hpal and then ligated to a linker molecule containing two EcoRI sites in tandem. After 
heat inactivation of the ligase enzyme the DNA was digested with EcoRI and a 1200 bp fragment isolated. 
This DNA fragment was then treated with Hpall and a 464 bp fragment 21 isolated. A plasmid 22 containing 
56 cDNA prepared from human growth hormone mRNA was prepared as described by Martial et aT "Science" 
205 : 602-606 (1979) and Roskem et al., "Nucleic Acids Res." 7: 305-320 (1979) (see~~a]so European 
Publication No. 127305). Plasmid 22 was digested with Hpall and a~461 bp fragment was isolated. The 461 
bp fragment was further digested with PstI and a 200 bp fragment 23 containing part of the hGH gene then 



15 



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isolated. DNA fragments 21 and 23 were ligated to a 3609bp DNA fragment isolated from pBR322 that had 
been previously digested"with EcoRI and Pstl. This DNA ligation mixture was used to transform E. coli 294 
to tetracycline resistance. A plasmid 24, designated pcHGHpps, was recovered from a transformant colony. 
In plasmid 24 the gene encodlrig'the AP promoter and signal sequence in pcHGHpps was linked to 

6 hGH in the sami' reading frame. However a number of extraneous nucleotides were present between the 
signal sequence and the beginning of the mjature hGH gene. This extraneous nucleotide sequence was 
deleted by mutagenesis. pcHGHpps was digested with EcoRI and Pstl and the 663 bp fragment 25 isolated. 
Fragment 25 was introduced into M13mp9 previously digested with Pstl and EcoRI. This was ligated and 
used to transform E. coli JM101. Clear plaques were selected and a derivative phage 26, M13mp9- 

70 cHGHpps, was identified~and isolated. Phage 26 was annealed to the synthetic oligonucleotide primer 
5'PCTGTGACAAAAGCCTTCCCAACCATTCC-OHT. The first 14 nucleotides correspond to the sequence of 
the 3' end of the.AP signal peptide and the last 14 correspond to the 5' end of the mature hGH coding 
sequence. The site-specific deletion mutagenesis was performed as previously described in Example 3 (see 
also J. Adelman et at. op cit.). Plaques containing the desired deletion were detected by Southern analysis 

75 with the 5'-32P-labeled oligonucleotide primer of this Example. Without enrichment for the desired 
genotype, nine percent of the plaques screened hybridized to the labelled primer. One of the positives, 
M13mp9-AP-1, 27, was determined by the dideoxy chain termination nucleotide sequencing method to have 
the expected sequence. The partial hGH gene, now correctly fused to the AP promoter and signal gene, 
was introduced into pHGH207, 28 (H. de Boer et al., op cit). This was accomplished by digesting pHGH207 

20 with Pstl and Ndel, and then isolating the 2750'bp~fragment. Another sample of pHGH207 was digested with 
Ndel and EcoRI and a 2064 bp fragment isolated. The M13mp9-AP-1 phage was digested by EcoRI and 
Pstl, and the 602bp AP hGH partial gene 29 was isolated. The 2750 and 2064 bp fragments were ligated in 
a three-part ligation with fragment 29, and the ligation mixture then used to transform E. coli 294 to 
ampicillin resistance. pAP-1 was isolated from a resistant colony and characterized by restriction enzyme 

26 mapping and nucleotide sequence analysis. N.B. This Is not an embodiment of the invention. 

Example 6 

Construction of a Plasmid (pAP-STII-hGH) to Express and Secrete hGH Under the Control of the AP 
30 Promoter 

ptrp-STII-hGH (from Example 3) was digested with Hpal and EcoRI and the vector fragment 31 isolated. 
A 420 bp AP promoter fragment 32 was isolated from the plasmid pAP-1 after digestion with "EcoRI and 
partial digestion with Rsal. Fragments 31 and 32 were ligated and used to transform E. coli 294 to ampiclNin 
35 resistance. The plasmid pAP-STII-hGH was isolated and characterized by restriction enzyme mapping and 
nucleotide sequence analysis. The nucleotide sequence and translated amino acid sequence of the AP-STII- 
hGH construction is shown in figure 5. 

Example 7 

40 

Recovery of hGH from E. coli Containing the Plasmid pAP-STII-hGH. 

E. coli W3110 and 294 were transformed respectively with pAP-STII-hGH or pAP-1 and cultured as 
described In Example 4 except that the medium used was phosphate depleted. The amounts synthesized 
45 and the distribution of processed and unprocessed hGH were determined as described in Example 4. The 
results are described in Table 1. In small volumes ptrp-STll-hGH produces better results, as can be seen 
from Table 1, but in 10 liter culture volumes the preferred embodiment is the plasmid pAP-STII-hGH since 
AP promoted cells can grow to higher densities than trp promoted organisms. 

50 Example 8 

Large Volume Fermentation and hGH Recovery 

Eight hours prior to the start of a 10 liter fermentation a 500 ml inoculum culture is grown up, A 
65 transformant of E. colt W3110 (tonA, phoA. phoT) (Example 9) containing pAP-STII-hGH is inoculated into a 
sterile 2 liter flask containing 500 ml of LB medium and tetracycline (0.5 ug/ml). The culture Is incubated In 
a rotary shaker at 37 'C for eight hours. A sterile 10 liter fermentation medium Is prepared, containing the 
following ingredients: 26g K2HPO4. 13g NaH2P04 2H20, 15g KCI, 50g (NH4)2S04. lOg Naa citrate, 50 ml of 



16 



EP 0 177 343 B1 



50 percent glucose, 1000 ml of 10 percent NZ-amine YT, 100 ml of 1M MgSO*, 5 ml of 2.7 percent FeCIa, 
5 ml of trace metals, 1 ml of 5 mg/ml tetracycline, 5 ml of antifoaming agent, and 6.5 liters of H2O. The 
starting pH of the medium is titrated to 7.5 by adding H2SO4, and the run is begun by seeding the 500 ml 
inoculum culture into the 10 liter fermenter. Throughout the run the temperature is maintained at 37*C and 
5 the culture agitated under aeration. From the outset, the cells are fed glucose (50 percent) at a flow rate of 

0. 5 ml/min. When the OD 550 is in the range 10-25 the glucose feed rate is manually adjusted to keep the 
pH at 7.5 and the residual glucose level ^ 1/4 percent. When the OD 550 reaches 25, the glucose feed rate 
is manually adjusted to drive the 6O2 level to 30 percent and thereafter, the glucose feed rate is periodically 
adjusted to maintain the d02 level at 30 percent. Thirty-six hours after the start of fermentation the cells are 

70 killed and harvested. 

The glucose feed and aeration are turned off but the agitation rate of 650 rpm is maintained. 1-butanol 
is added immediately to the fermenter to yield a final concentration of 1.5 percent and steam is immediately 
injected into the fermenter jacket so that the temperature in the tank rises rapidly to 50 "C. When the 
temperature reaches 50 "C. it is held at this temperature for 10 minutes. Then the fermenter is rapidly 

75 cooled below 20 and the cellular contents of the fermenter are harvested by centrifugation. The cell 
paste is first frozen at -20* C and then transferred to a -80* C freezer. 

The cell paste, frozen at -80 'C, is thawed overnight at 4''C and all subsequent steps are performed at 
4*C. The. paste is mixed in 4 volumes of lOmM Tris-HCI.pH = 8.0 and suspended in an Ultra-Turrex 
homogenizer for 30 seconds. The suspension is stirred for 30 minutes and then the cells are removed by 

20 centrifugation at 12,000 x g for 30 minutes. The periplasmic proteins contain in the supernatant mature hGH 
at between 0.5 and 1 gram/liter/100 OD550 with about 95 percent of the total hGH (as estimated from 
immunoblots with peroxidase-coupted anti-hGH) in processed form in the periplasm. About 50-60 percent of 
the total cellular hGH is recovered in the supernatant. The supernatant contairis about 20 percent hGH by 
weight of protein. 

26 

Example 9 

Construction of a Host Organism W3110 tonA, phoA, phoT 

30 The host organism for the fermentation was constructed in several steps using standard techniques 
involving transductions with phage derived from Pi (J. Miller, Experiments in Molecular Genetics). The phoT 
and phoA mutations were sequentially cotransduced from E. coli into strain W3110 tonA with the aid of 
genetically linked antibiotic-resistance transposons. The presence of the phoT mutation, which was intro- 
duced first, was recognized since these transductants form blue colonies on high phosphate, 

35 phosphochromogen-containing plates (5-bromo-4-chloro-3-indoylphosphate).The introduction of the phoA 
mutation was recognized as transductants which form white colonies on low phosphate, phosphochromogen 
plates. This phoT mutant, or a phoR mutant constructed in similar fashion, when transformed with pAP-STII- 
hGH, secretes over 90 percent of the total hGH into the periplasmic space over the course of the 
fermentation. In contrast, a non-constitutive bacterium such as W3110 only secretes about 50 percent of the 

40 hGH into the periplasm and total expression levels are somewhat lower in some cases. The presence or 
absence of the phoA mutant makes little or no difference in the yield of heterologous protein. However, 
since phoA mutants do not secrete AP, it is not necessary to separate AP in the course of purifying 
periplasmic hGH from phoA mutants. 

45 Claims 

1. A hybrid DNA sequence encoding a protein having at least the amino terminal sequence of mature 
hGH operably linked to a DNA sequence encoding an enterotoxin signal. 

50 2. A hybrid DNA sequence according to claim 1 wherein.the signal is the STII signal. 

3. A replicable vector containing, in 5* to 3' order, a promoter, a Shine-Dalgarno sequence, a hybrid DNA 
sequence according to claim 1 or claim 2, and a termination region comprising a stop codon; said 
promoter controlling transcription of the hybrid DNA sequence. 

4, A method comprising; 

(a) constructing a vector comprising a DNA sequence encoding a protein having at least the amino 
terminal sequence of mature hGH operably ligated to a DNA sequence encoding an enterotoxin 



17 



EP 0 177 343 B1 



signal, the DNA being operabiy linked to the STII Shine-Dalgarno sequence; 

(b) transforming a host cell capable of processing the hybrid and secreting the mature eukaryotic 
protein with the vector; 

(c) culturing the transformed host cell; and 

5 (d) recovering the mature protein which comprises at least the amino terminal sequence of hGH 

from the periplasm of the cell. 

5. A prokaryotic cell culture comprising; 

(a) a mature eukaryotic protein and 

10 (b) a hybrid DNA encoding a direct fusion protein of an enterotoxin signal directly fused at its 

carboxyl terminus to the amino terminus of the mature eukaryotic protein; and wherein the mature 
protein is located in the periplasm of the cell. 

6. A prokaryotic cell culture comprising; 
75 (a) mature hGH and 

(b) a hybrid DNA encoding a direct fusion protein of an enterotoxin signal directly fused at its 
carboxyl terminus to the amino terminus of mature hGH; and wherein the mature protein is located 
in the periplasm of the cell. 

20 7. A cell culture according to claim 5 or 6 wherein greater than about 25% of the total weight of mature 
and fusion protein is located in the cell periplasm. 

8. The culture of claim 5, 6 or 7 wherein the total weight of mature and fusion protein is greater than about 
2mg/litre of culture on a culture optical density basis of 1 at 550 nm and greater than 50 percent of the 

25 mature protein by weight is present in the periplasm. 

9. The culture of claim 8 wherein said total weight is greater than 4mg/litre. 

10. The culture of claim 8 or 9 wherein greater than 80% of the mature protein by weight is present in the 
30 periplasm. 

11. A protein comprising an enterotoxin signal directly fused at its carboxyl terminus to the amino terminus 
of a mature eukaryotic protein. 

35 12. A method according to claim 4 wherein said step of recovering the mature protein from the periplasmic 
space comprises: 

(a) killing the cell; 

(b) freezing the cell; 

(c) thawing the cell; and 

40 (d) separating the periplasmic proteins, including the eukaryotic protein, from the remainder of the 

cells. 

13. The method of claim 12 wherein the cells are killed by 

(a) contacting the cell with an alkanol which is not lytic for cell membranes, and; 
45 (b) heating the cell. 

14, A method of claim 13 wherein the cell is cultured in a phosphate-limiting culture medium prior to 
freezing the cell. 

60 15. A method for the recovery of mature hGH from a bacterial cell comprising carrying out a method 
according to claim 6 wherein said DNA sequence encodes the full sequence of mature hGH; and 
wherein said recovery step (d) comprises: 

(e) killing the cell; 

(f) freezing the cell; and 

55 (g) separating periplasmic protein from the remainder of the cell. 

Revendlcations 



18 



EP 0 177 343 B1 



1. Sequence d'ADN hybride codant pour une proteine ayant au moins la sequence amino-ternninale de la 
hGH mature li^e de maniere fonctionnelle a une sequence d'ADN codant pour un signal d'ent^rotoxine. 

2. Sequence d'ADN hybride suivant la revendlcation 1 , dans laquelle le signal est le signal STII. 

5 

3. Vecteur d*ADN r^plicable contenant, dans le sens 5* h Z\ un pronnoteur, une sequence Shine-Dalgarno, 
une sequence d'ADN hybride suivant la revendicatlon 1 ou la revendication 2. et une region de 
terminaison comprenant un codon d*arret ; ledit promoteur r^gulant la transcription de la sequence 
d'ADN hybride. 

70 

4. Proc^d^ consistant : 

(a) a construire un vecteur connprenant une sequence d'ADN codant pour une proteine ayant au 
nnoins la sequence anrti no-term inale de la hGH mature r^unie par ligation fonctionnelle h une 
sequence d'ADN codant pour un signal d'enterotoxine, I'ADN 6tant \\6 de maniere fonctionnelle a la 

75 sequence Shine-Dalgarno de STII ; 

(b) a transformer une cellule-hote capable d'effectuer la maturation de I'hybride et de secreter la 
proteine eucaryotique mature avec le vecteur ; 

(c) Si cultlver la cellule-hote transform^e ; et 

(d) a recueillir la proteine mature qui comprend au moins la sequence amino-terminale de la hGH a 
20 partir du p^riplasme de la cellule. 

5. Culture de cellules procaryotiques, comprenant : 

(a) une proteine eucaryotique mature, et 

(b) un ADN hybride codant pour une proteine de fusion directe d'un signal d'enterotoxine fusionn^ 
25 directement a son extr^mit^ carboxyle a Textr^mit^ amino de la proteine eucaryotique mature ; la 

proteine mature etant presente dans le periplasme de la cellule. 

6. Culture de cellules procaryotiques comprenant : 

(a) la hGH mature, et 

30 (b) un ADN hybride codant pour une proteine de fusion directe d'un signal d'enterotoxine fusionn^ 

directement a son extremite carboxyle a I'extr^mite amino de la hGH mature ; la proteine mature 
etant presente dans le p§riplasme de la cellule. 

7. Culture de cellules suivant la revendication 5 ou 6, dans laquelle une quantity sup^rieure ^ environ 
35 25% du poids total de la proteine mature et de fusion est presente dans le periplasme de la cellule. 

8. Culture suivant la revendication 5, 6 ou 7, dans laquelle le poids total de la proteine mature et de fusion 
est superieur a environ 2 mg/litre de culture, sur la base d'une density optique de la culture de 1 a 550 
nm, et plus de 50% de la proteine mature, en poids, sont presents dans le periplasme. 

40 

9. Culture suivant la revendication 8, dans laquelle le poids total est superieur a 4 mg/litre. 

10. Culture suivant la revendication 8 ou 9, dans laquelle plus de 80% de la proteine mature, en poids, sont 
presents dans le periplasme. 

. 45 

11. Proteine comprenant un signal d'enterotoxine fusionne directement a son extremite carboxyle a 
I'extremite amino d'une proteine eucaryotique mature. 

12. Procede suivant la revendication 4, dans lequel I'etape de separation de la proteine mature de I'espace 
50 p^riplasmique consiste : . 

(a) a tuer la cellule ; 

(b) ^ congeler la cellule ; 

(c) a d^congeler la cellule ; et 

(d) a separer les prot^ines periplasmiques, comprenant la proteine eucaryotique, du reste des 
55 cellules. 

13. Proc6de suivant la revendication 12, dans lequel les cellules sont tu^es 

(a) par mise en contact de la cellule avec un alcanol qui ne possede pas d'action de lyse sur les 



19 



EP 0 177 343 B1 



membranes cellulaires, et 

(b) par chauffage de la cellule. 

14. Proc^d4 suivant la revendication 13. dans lequel la cellule est cultiv^e dans un milieu de culture 
5 limitant en phosphate avant congelation de la cellule. 

15. Precede pour separer de la hGH mature d'une cellule bact^rienne, consistant a mettre en oeuvre un 
precede suivant la revendication 6, dans lequel la sequence d'ADN code pour la sequence totale de la 
hGH mature ; et dans lequel Tetape de separation (d) conslste : 

70 (e) a tuer la cellule ; 

(f ) a congeler la cellule ; et 

(g) a separer la proteine periplasmique du teste de la cellule. 
PatentansprUche 

75 

1. Hybrid-DNA-Sequenz, die fiir ein Protein kodiert, bei dem zumindest die Aminoterminalsequenz von 
reifem hGH mit einer DNA-Sequenz operabel verbunden ist, die fiir ein Enterotoxinsignal kodiert. 

2. Hybrid-DNA-Sequenz nach Anspruch 1 , worin das Signal das STII-Signal ist. 

20 

3. Replizierbarer Vektor, der in 5' bis 3* Reihenfolge einen Promoter, eine Shine-Dalgarno-Sequenz. eine 
Hybrid-DNA-Sequenz nach Anspruch 1 bder 2 und einen Terminationsbereich aufweist, der ein 
Stopcodon umfaflt; wobei der genannte Promoter die Transkription der Hybrid-DNA-Sequenz steuert. 

25 4, Verfahren, umfassend: 

(a) das Konstruieren eines Vektors, der eine. DNA-Sequenz umfaflt, die fur ein Protein kodiert, bei 
dem zumindest die Aminoterminalsequenz von reifem hGH mit einer DNA-Sequenz operabel ligiert 
ist, die fur ein Enterotoxinsignal kodiert. wobei die DNA operabel mit der STII-Shine-Dalgarnose- 
quenz verbunden ist; 

30 (b) das Transformieren einer Wirtszelte, die fahig ist, das Hybrid zu verarbeiten und das reife 

eukaryotische Protein mit dem Vektor auszuscheiden; 

(c) das Kultivieren der transform ierten Wirtszelle; und 

(d) das Wiedergewinnen des reifen Proteins, das zumindest die aminoterminale Sequenz von hGH 
umfaiBt, aus dem Periplasma der Zelle. 

35 

5. Prokaryotische Zellkultur, umfassend: 

(a) ein reifes eukaryotisches Protein und 

(b) eine Hybrid-DNA, die fur ein direktes Fusionsprotein eines Enterotoxinsignals kodiert, das an 
seinem Carboxylterminus direkt mit dem Aminoterminus des reifen eukaryotischen Proteins ver- 

40 schmolzen ist; und worin das reife Protein sich im Periplasma der Zelle befindet. 

6. Prokaryotische Zellkultur, umfassend: 

(a) reifes hGH und 

(b) eine Hybrid-DNA, die fur ein direktes Fusionsprotein eines Enterotoxinsignals kodiert, das an 
45 seinem Carboxylterminus direkt mit dem Aminoterminus von reifem hGH verschmolzen ist; und 

worin das reife Protein sich im Periplasma der Zelle befindet. 

7. Zellkultur nach Anspruch 5 Oder 6, worin sich mehr als etwa 25% des Gesamtgewichts an reifem und 
Fusionsprotein im Zellperiplasma befinden. 

60 

8. Kultur nach einem der Anspruche 5, 6 Oder 7 , worin das Gesamtgewicht an reifem und Fusionsprotein 
groBer als etwa 2mg/Liter Kultur auf einer optischen Dichtebasis der Kultur von 1 bei 550 nm ist, und 
mehr als 50 Gew.-% des reifen Proteins im Periplasma vorhanden sind. 

56 9. Kultur nach Anspruch 8, worin das genannte Gesamtgewicht grofier als 4 mg/Liter ist. 

10. Kultur nach Anspruch 8 Oder 9, worin mehr als 80 Gew.-% des reifen Proteins im Periplasma 
vorhanden sind. 



20 



EP 0 177 343 B1 



11. Protein, das ein Enterotoxinsignal umfajSt, das an seinem Carboxylterminus direkt mit dem Aminotermi- 
nus eines reifen eukaryotischen Proteins verschmolzen ist. 

12. Verfahren nach Anspruch 4, worin der genannte Schritt des Wiedergewinnens des reifen Proteins vom 
5 periplasmischen Raum umfaJSt: 

(a) das Abtoten der Zelle; 

(b) das Einfrieren der Zelle; 

(c) das Auftauen der Zelle; und 

(d) das Abtrennen des periplasmischen Proteins, einschliefllich des eukaryotischen Proteins, vom 
70 Rest der Zellen. 

13. Verfahren nach Anspruch 12, worin die Zellen abgetotet werden durch 

(a) In-Kontakt-bringen der Zelle mit einem Alkanol, das nicht lytisch fur Zellmembranen ist, und 

(b) Erwarmen der Zelle. 

14. Verfahren nach Anspruch 13, worin die Zelle vor dem Einfrieren der Zelle in einem phosphatbeschran- 
kenden Kulturmedium kultlviert wird. 



15. Verfahren zur Wiedergewinnung von reifem hGH aus einer bakteriellen Zelle, welches die Durchfuhrung 
20 eines Verfahrens nach Anspruch 6 umfaiSt, worin die genannte DNA-Sequenz fUr die voile Sequenz von 
reifem hGH kodiert; und worin der genannte Wiedergewinnungsschritt 

(d) umfai5t: 

(e) das Abtuten der Zelle 

(f) das Einfrieren der Zelle; und 

25 (g) das Abtrennen des periplasmischen Proteins vom Rest der Zelle. 



30 



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AP- hGH gene fusion 
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33 



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