Gene, 39 (1985) 247-254 Elsevier 241 GENE 1447 Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable (Recombinant DNA; plasmid vector; secretion; alkaline phosphatase; disulfide bond) Gregory L. Gray*, Jane S. Baldridge, Kathleen S. McKeown, Herbert L. Heyneker* and Chung Nan Chang Genentech, Inc., 460 Point San Bruno Boulevard, South Francisco, CA 94080 (U.S.A.) Tel. (415) 952-1000 (Received May 25th, 1985) (Revision received August lst, 1985) (Accepted August lOth, 1985) SUMMARY We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disultide bonds as authentic hGH. * Present address (G.L.G., to whom correspondence and reprint requests should be addressed, and H.L.H.): Genencor, Inc., 180 Kimball Way, South San Francisco, CA 94080 (U.S.A.) Tel. (415) 588-3475. Abbreviations: Ap, ampicillin; APase, alkaline phosphatase; BGal, /?-galactosidase; bp, base pair(s); cys, cysteine; DTT, dithiothreitol; hGH, human growth hormone; IFN, interferon; IPTG, isopropyl-fi-D-thiogalactopyranoside; LB, Luria broth; met-hGH, methionyl hGH; N, amino end; PA, polyacrylamide; pre-hGH, precursor of hGH; pho-hGH, hGH fused to the N-terminal signal peptide of APase; a, resistant; ‘, sensitive; SDS, sodium dodecyl sulfate; Tc, tetracycline; [ 1, designates plasmid-carrier state. INTRODUCTION The mechanism by which eukaryotic and pro- karyotic cells transport proteins has been conserved through evolution (Michaelis and Beckwith, 1982). Perhaps the most dramatic evidence of this has been the demonstration that prokaryotic secretory pro- teins can be exported and processed in eukaryotic cells (Roggenkamp et al., 1981), and that eukaryotic secretory proteins can be exported and processed in bacteria (Talmadge et al., 1980; Palva et al., 1983). There have been two reports of the transport of eukaryotic secretory proteins across the inner 0378-I 119/85/$03.30 0 1985 Elsevier Science Publishers
Gene, 39 (1985) 247-254
Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable
Gregory L. Gray*, Jane S. Baldridge, Kathleen S. McKeown, Herbert L. Heyneker* and Chung Nan Chang
Genentech, Inc., 460 Point San Bruno Boulevard, South Francisco, CA 94080 (U.S.A.) Tel. (415) 952-1000
(Received May 25th, 1985)
(Revision received August lst, 1985)
(Accepted August lOth, 1985)
We have studied the synthesis, secretion, and processing of human growth hormone (hGH) in Escherichia
coli transformed with plasmids engineered for the expression of hGH as a secreted product. In one plasmid, pPreHGH207-2, the coding sequence of the natural hGH precursor (pre-hGH) is placed under the control of
the E. coli trp promoter. In a second plasmid, pAPH-1, a DNA fragment containing the E. coli alkaline phosphatase promoter and signal sequence codons is fused to the mature hGH coding sequence (pho-hGH). Most of the hGH was present in the osmotic shock fluids of E. coli cells containing either plasmid, indicating transport to the periplasmic space. Amino acid sequencing of the N termini of the pre-hGH and pho-hGH gene products revealed that both were processed correctly. Electrophoretic analysis of these polypeptides on reducing and nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels indicates that periplasmic hGH is monomeric and contains the same two disultide bonds as authentic hGH.
* Present address (G.L.G., to whom correspondence and reprint
requests should be addressed, and H.L.H.): Genencor, Inc., 180
Kimball Way, South San Francisco, CA 94080 (U.S.A.)
The mechanism by which eukaryotic and pro- karyotic cells transport proteins has been conserved through evolution (Michaelis and Beckwith, 1982). Perhaps the most dramatic evidence of this has been the demonstration that prokaryotic secretory pro- teins can be exported and processed in eukaryotic cells (Roggenkamp et al., 1981), and that eukaryotic secretory proteins can be exported and processed in bacteria (Talmadge et al., 1980; Palva et al., 1983). There have been two reports of the transport of eukaryotic secretory proteins across the inner
membrane of E. coli. Chicken ovalbumin is syn- thesized on membrane-bound polysomes and se- creted into the E. coli periplasmic space (Fraser and Bruce, 1978; Baty et al., 1981). However, as in the natural host, its mode of secretion is unusual because it occurs without cleavage of an N-terminal signal peptide. There is only one well-documented case in which a eukaryotic protein has been both cleaved from a signal peptide containing precursor and transported to the E. coli periplasm. Talmadge et al. (1980) showed that proinsulin is correctly processed and transported to the bacterial periplasm. They showed that transport also occurred when DNA corresponding to the codons for the signal peptide and the first few amino acids of the mature form of pBR322-encoded b-lactamase was used to replace the DNA for the natural signal peptide. Processing occurred at the /J-lactamase cleavage site, yielding a P-lactamase-proinsulin fusion protein.
In this paper we describe the synthesis, secretion and correct processing in E. coli of hGH. Expression vectors were constructed encoding either the natural hGH precursor or a hybrid precursor in which the codons for the E. cob alkaline phosphatase (phoA) signal peptide and the natural form of hGH are precisely fused. In addition, evidence that periplas- mic hGH contains the same two disulfide bridges as authentic hGH is presented. This result suggests that the mechanisms within the secretory pathways of both prokaryotes and eukaryotes for the ordered formation of disulfide bonds of proteins may be similar.
MATERIALS AND METHODS
(a) Bacterial strains, plasmids, and growth con-
E. coli 294 (Goeddel et al., 1979) was used for all experiments. Plasmids pHGH507, pPreHGH207-2 and a pHGHcDNA (Gray et al., 1984a), pHGH- 207-l (De Boer et al., 1982), and PHI-1 (Inouye et al., 1981) have been described. For hGH expres- sion experiments cells were grown overnight at 37’ C in medium supplemented with 20 pg Ap/ml. LB medium was used for growth of all strains, except for
294[pAPH-11, which was grown in low-phosphate medium (Inouye et al., 198 1). 1 mM IPTG was used to induce the cytoplasmic enzyme PGal.
(b) Electrophoretic analysis of hCH
The hGH from the 294[pPreHGH207-21 or 294[ pAPH 1 ] cells was obtained from osmotic-shock fluids. The proteins in the fluids were concentrated by lyophilization and then subjected to immuno- affinity chromatography on a column containing monoclonal anti-hGH coupled to Affigel 10 (BioRad). hGH containing fractions were pooled. Aliquots were denatured by boiling in SDS in the presence or absence of DTT as previously described (Pollitt and Zalkin, 1983). The proteins were then separated on a 12.5% PA-SDS gel (Pollitt and Zalkin, 1983), followed by staining with Coomassie blue. The hGH produced in the 294[pHGH507] culture was detected by an immunoblot method as follows (Burnette, 1981). Proteins from sonically disrupted cells (10 bursts of 2 s) were prepared and separated as described above. Following electro- phoresis, the proteins were transferred to nitro- cellulose paper. After incubation in 1% gelatin, the paper was treated with polyvalent rabbit anti-hGH serum (1: 100 dilution). The antigen-antibody com- plexes were detected with i2?-labeled protein A and autoradiography. The met-hGH standard was pre- pared from E. coli (Jones and O’Connor, 1982).
(c) Amino acid sequence analysis
The immunoaffinity-purified hGH preparations from 294[ pPreHGH207-21 or 294[ pAPH- 1 ] OS-
motic-shock fluids described above were subjected to automated N-terminal amino acid sequencing (Edman and Begg, 1967).
RESULTS AND DISCUSSION
(a) Expression of hGH in E. coli
The previously described plasmid pPreHGH207-2 (Gray et al., 1984a) contains the coding region for the natural precursor form of hGH (pre-hGH) placed just downstream from the E. coli trp promo-
ter. It was derived from pHGH207-1 by interposing the hGH signal peptide coding region (Fig. la) between the promoter and the codons corresponding to the mature hGH polypeptide. Table I shows that 294[pPreHGH207-21 cells grown in rich medium (LB) produce hGH at a level of 600 ng/ml/A,,,.
In plasmid pAPH-1 (construction detailed in Fig. lb) a DNA segment comprising the E. cofiphoA promoter and signal peptide codons (Fig. la) is precisely fused to the codons for mature hGH(pho-hGH). In 294[pAPH-l] cells grown in
low-phosphate medium, a growth condition which results in the derepression of the APase promoter (Inouye et al., 1981), hGH-related material is pro- duced at a level of 280 “g/ml/A,,, (Table I). Addi- tion of excess inorganic phosphate reduces hGH accumulation over IOO-fold (not shown). The ratio of hGH levels under derepressing and repressing con- ditions appears to approach that of APase (200- 300-fold). This suggests that in 294[pAPH-l ] cells hGH production, like that of APase itself, may be controlled at the level of transcription initiation.
Compartmentalization of hGH, plasmid-derived /I-lactamase, and /IGal in stationary-phase E. colt’ 294 cultures
Except for 294[pAPH-I], cells were grown overnight in LB medium supplemented with 20 pg Ap/ml and 1 mM IPTG. 294[pAPH-I]
cells were grown overnight in low-phosphate medium (Inouye et al., 1981) supplemented with 20 pg Ap/ml and 1 mM IPTG. A constant
number of cells (1 ml adjusted to Asso = 1.0) were fractionated by the osmotic-shock method, exactly as described by Koshland and
Botstein (1980). The effectiveness of the procedure was monitored by the measurement of the periplasmic marker enzyme /?-lactamase
and the cytoplasmic marker enzyme /?Gal. /I-Lactamase was measured calorimetrically by hydrolysis of 7-(thienyl-2-acetamido)-
3[2(4-N,N-dimethylamino-phenylazo)pyridinium methyl]-3-cephem-4-carboxylic acid (Jones et al., 1982). /IGal was measured by
hydrolysis of O-nitrophenyl-fl-D-galactopyranoside (Miller, 1972). hGH was assayed by RIA (Goeddel et al., 1979).
Plasmid hGH gene expressed Fraction hGH by RIA p-lactamase BGal
(% of total) (% of total)
(ng/mV&a) (% of total)
pBR322 none supernatant
total in culture
(1 - 22 1
<l - 4 0
<l - 68 3
<l - 6 96
<4 - 100 100
supernatant 190 2 17 1
cell wash 15 0 10 2
shock fluid 1800 17 73 3
shocked cells 8 700 81 0 94
total in culture 11000 100 100 100
supernatant 7 1 17 2
cell wash 3 0 21 2
shock fluid 450 76 58 1
shocked cells 140 23 4 95
total in culture 600 100 100 100
pAPH-1 pho-hGH supernatant 1 1 12 4
cell wash 34 12 17 6
shock fluid 230 82 64 9
shocked cells 15 5 7 81
total in culture 280 100 100 100
met ala tfw 9ty SM or9 thr SW Iw leu IOU o,o @a 9/v Iw Ia/ cys lsu prv rrp /eu 9,” 9/u 9/v se, ,,I.,
nx?, lye 9/n SW thr i/e olo /eu o/o /eu ,a/ pro leu let, 9he thr pro vol Ihr lys o/o phoA: 5’.. .GUG AAA CPA AGC ACU AUU GCA CUG GCU GUC UUA CCG UUA CUG UUU ACC CCU GUG ACA ABA GCC 3’
T4 DNA I,gr,se + ATP EC&, DNA Pal I I large fragment I + dNTPs
Ikolote 2064 bp frogmenf I I Al I h’de I Isolate -2750bp fragment
1 T4 DNA Ingase
Fig. 1. Construction of precursor hGH vectors. (a) The mRNA and amino acid sequences for the signal peptide of hGH and alkaline
phosphatase. (b) The strategy for the construction ofpAPH-I. Plasmid PHI-1 was used to derive a 464-bp EcoRI-HpaII DNA segment
bearing thephoA promoter and signal peptide codons (Inouye et al., 1981; Kikuchi et al., 1981). Plasmid pcHGHpps and the Ml3 phage
mp9HGHppsUF contain this DNA fragment placed upstream of a segment of DNA containing codons for the 3’ region of the hGH
(b) Cellular localization of bGH
Stationary-phase cells were fractionated by the osmotic-shock method, which releases secreted periplasmic proteins without cell lysis (Koshland and Botstein, 1980). To demonstrate the efficacy of the procedure the segregation of the periplasmic enzyme /I-lactamase and the cytoplasmic enzyme /I-Gal was monitored. Another control to monitor possible cell lysis was to follow the segregation of met-hGH in 294[pHGH507] cells which express hGH as a cytoplasmic product (Gray et al., 1984a).
The results (Table I) show that in 294[pPreHGH- 207-21 cells 76 y0 of the hGH is found in the osmotic- shock fluid and thus most of it appears to be secreted across the inner membrane into the periplasmic space. In 294[pAPH-l] cells grown in low phos- phate medium the hGH was also localized to the periplasmic fraction (82%). Thus the use of a bac- terial leader peptide also appeared to effect the periplasmic transport of the hGH polypeptide effi- ciently.
(c) Processing of periplasmic hGH
The hGH-related proteins present in the osmotic shock fluids from 294[pPreHGH207-21 and 294[pAPH- l] cells were purified to homogeneity by a one-step procedure employing immunoaflinity chromatography (see MATERIALS AND METHODS,
section b). Less than 10% of the hGH was lost as a result of the purification procedure. The electro- phoretogram of the reduced purified protein is shown in Fig. 2, lanes l-3. The hGH derived from the osmotic-shock fluids of either 294[pPreHGH- 207-21 or 294[pAPH-l] cells has the same mobility
hGH (red) hGH (nonred)
Fig. 2. Comparison
I 1 I I + OTT - DTT
of periplasmic hGH to met-hGH. Purified
met-hGH (Jones and O’Connor, 1982) and immunoaffkity-
purified hGH extracted from cells containing the pre-hGH
plasmid pPreHGH207-2 or the pho-hGH plasmid pAPH-1 was
resolved on a 12.5% PA-SDS gel. The mobilities of proteins in
their reduced states (left lanes) are compared with those in their
nonreduced states (right lanes). The positions of reduced and
nonreduced hGH are shown (Jones and O’Connor, 1982).
as the reduced met-hGH standard, indicating that the precursor proteins were processed to a size very similar to met-hGH. The purified periplasmic hGH preparations were subjected to N-terminal amino acid sequencing by the Edman degradation method (Edman and Begg, 1967). In both cases the only sequence observed was phe-pro-thr-ile, which is identical to that of authentic (mature) hGH.
signal peptide and the 5’ region of the mature form of hGH. In mp9APH-1 the nucleotides between the phoA signal peptide codons
and the N-terminal mature hGH codons have been deleted by site-specific mutagenesis. In pAPH-1 the fused sequence containing the
phoA promoter and signal peptide codons precisely juxtaposed to the N-terminal mature hGH codons replaces the trp promoter and
N-terminal mature hGH codons of pHGH207-1. The site-specific deletion mutagenesis was performed as previously described (Adelman
et al., 1983) except that only one oligonucleotide was used to prime second-strand synthesis on the single-stranded Ml3 phage template.
The oligonucleotide synthesized was 5’-CTGTGACAAAAGCCTTCCCAACCATTCC-3’. The first 14 nucleotides correspond to the
3’ end of the phoA signal peptide codons and the last 14 nucleotides correspond to the 5’ end of the mature hGH codons. Plaques
containing the desired deletion were detected by hybridization with the 5’-32P-labeled oligonucleotide primer as previously described
(Adelman et al., 1983). The expected DNA sequence of one of the positives, mp9APH-I, was confirmed by the dideoxy chain termination
method (Sanger et al., 1977). Symbols: solid boxes, antibiotic resistance genes; open boxes, APase or hGH promoters or mature
polypeptide coding regions; stippled boxes, hGH signal peptide codons; cross-hatched boxes, APase signal peptide codons; Paho, APase
promoter, P,,,, E. co/i tryptophan operon promoter; single-headed arrows indicate direction of transcription; double-headed arrows
indicate DNA fragment isolated.
(d) Electrophoretic analysis of periplasmic hGH
The cytoplasmic met-hGH in extracts of 294[pHGH507] cells displays a multitude of electro- phoretic forms, including monomers with differing disulfide bridges as well as dimers and higher order oligomers, when the extract proteins are not first reduced (Fig. 3, lanes 1,3). We postulate that the generation of these forms is a consequence of the sudden transfer of the met-hGH from the reducing environment of the bacterial cytoplasm (Pollitt and Zalkin, 1983) to a more oxidizing environment upon preparation of the cell extract. If the proteins in the cell extract are first reduced, the hGH is monomeric and has essentially a single mobility corresponding to that of the reduced met-hGH standard (Fig. 3, lanes 4,6). In contrast, periplasmic hGH is homo- geneous and monomeric in both its reduced and unreduced states. The mobilities of these states, which differ from each other, are similar to the
hGI hGH (red)
ibonred) - -
III - DTT + DTT
Fig. 3. Immunological analysis of hGH produced in the cyto-
plasm of cells containing the met-hGH expression plasmid
pHGH507. Whole cell extracts were boiled in the absence or
presence of DTT and the proteins analyzed by the “Western”
blotting procedure as described in MATERIALS AND
METHODS, section b. Purified met-hGH was used as a stand-
ard. Cells transformed with pBR322 were used as a negative
control. The positions of the reduced and unreduced forms of the
met-hGH standard are indicated.
corresponding states of the purified standard met- hGH or authentic hGH (Jones and O’Connor, 1982), which are known to possess the correct disulfide bridges (cys 53-cys 165 and cys 182- cys 189; Kohr et al., 1982).
(1) In Pseudomonas aerugznosa, hGH is trans- located across the inner membrane and correctly cleaved from its natural (pre-hGH) precursor (Gray et al., 1984a). The occurrence of the same phe- nomena in E. coli indicates that the mechanism involved is closely related in genetically unrelated Gram-negative bacteria. It is of interest that E. coli and P. aeruginosa do not process and transport all secreted proteins in the same way (Lory and Tai, 1983; Gray et al., 1984b).
(2) Although bacterial signal peptides in general have no sequence homology with each other, they share several distinct features which appear to be required for these processes: (i) one or more basic residues occur near the N terminus; (ii) the basis section is followed by a stretch of uncharged, mostly hydrophobic amino acids; and (iii) the C-terminal amino acid has a small side chain (Michaelis and Beckwith, 1982). Pre-hGH possesses a signal pep- tide with these characteristics (Fig. la) which may account for the efficient transport and correct pro- cessing of hGH by E. coli.
(3) ThephoA signal peptide functions efficiently in the hGH secretion process, although it has no homology to that of hGH (Fig. la). In another study, Palva et al. (1983) showed that the expression in Bacillus subtilis of a gene fusion containing the signal peptide codons of the a-amylase from Bacillus amyloliquefaciens attached by linkers to the mature coding sequence of human IFNcrD resulted in IFNolD transport and precise removal of the bac- terial signal peptide. The cleavage specificity resided in the signal peptide and was independent of which amino acid followed it. In addition, Talmadge et al. (1980) showed that, in E. coli, expression of a gene fusion containing codons for the signal peptide and the first few amino acids of the mature form of /?-lactamase connected by a linker to codons for rat proinsulin also resulted in proinsulin transport and cleavage of the precursor after the last amino acid of the signal peptide.
(4) In E. coli disultide-bond formation in the periplasmic protein pBR322-encoded /I-lactamase appears to take place when the protein is trans- located from the reducing environment of the cytoplasm to the perhaps more oxidizing environ- ment of the periplasm (Pollitt and Zalkin, 1983). Our results are consistent with this idea. The met-hGH in crude extracts of 294[pHGH507] cells is electro- phoretically very heterogeneous unless it is first sub- jected to complete reduction (Fig. 3). We believe that this heterogeneity reflects the complex disulfide chemistry of hGH which has been moved un- naturally from the (reduced) cytoplasm into a less reduced environment resulting from the electro- phoresis sample preparation. hGH under these cir- cumstances has the potential to form a variety of intrachain or interchain disultide bonds. In contrast, the hGH present in osmotic shock fluids, like authentic hGH, is monomeric and homogeneous in either its reduced or unreduced state. This suggests that periplasmic transport has a role in specifying the correct formation of disullide bridges in secreted proteins.
We thank Kim Dinkelspiel and Peter Ng for synthesis of oligonucleotides, John Bell and Henry Rodriguez for protein sequencing, Jeanne Arch and Alane Gray for help in manuscript preparation, and Andrew Jones for helpful discussions.
Adelman, J.P., Hayflick, J.S., Vasser, M. and Seeburg, P.H.: In
vitro deletional mutagenesis for bacterial production of the
20000-dalton form of human pituitary growth hormone.
DNA 2 (1983) 183-193.
Baty, D., Mercereau-Puijalon, O., Perrin, D., Kourilsky, P. and
Lazdunski, C.: Secretion into the bacterial periplasmic space
of chicken ovalbumin synthesized in Escherichia coli. Gene 16
Burnette, W.W.: “Western blotting”: electrophoretic transfer of
proteins from sodium dodecyl sulfate-polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with
antibody and radioiodinated protein A. Anal. Biochem. 112
De Boer, H.A., Comstock, L.J., Yansura, D.G. and Heyneker,
H.L.: Construction of a tandem trp-lac promoter for efficient
and controlled expression of human growth hormone gene in
Escherichia coli, in Rodriguez, R.L. and Chamberlin, M.J.
(Eds.), Promoters: Structure and Function. Praeger, New
York, 1982, pp. 462-481.
Edman, P. and Begg, G.: A protein sequenator. Eur. J. Biochem.
Fraser, T.H. and Bruce, B.J.: Chicken ovalbumin is synthesized
and secreted by Escherichiu coli. Proc. Natl. Acad. Sci. USA
75 (1978) 5936-5940.
Goeddel, D.V., Heyneker, H.L., Hozumi, T., Arentzen, R.,
Itakura, K., Yansura, D.G., Ross, M.J., Miozzari, G., Crea,
R. and Seeburg, P.H.: Direct expression in Escherichia coli of
a DNA sequence coding for human growth hormone. Nature
281 (1979) 544-548.
Gray, G.L., McKeown, K.A., Jones, A.J.S., Seeburg, P.H. and
Heyneker, H.L.: Pseudomonas aeruginosa secretes and cor-
rectly processes human growth hormone. Biotechnology 2