Secretion of correctly processed human growth hormone in E. coli and Pseudomonas

The production of mature hGH In E. coli and Pseudomonas strains transformed by a plasmid which encodes pre hGH (comprising the signal polypeptide and the hormone itself) is described. These prokaryotes process the pre-hGH to cleave the signal sequence and, thereby, produce mature hGH.

FIELD OF INVENTION 
This invention relates to the production of human growth hormone (hGH) with 
its signal peptide (pre-human growth hormone) in E. coli and Pseudomonas 
and processing of the unprocessed (pre) protein by the bacterial host to 
cleave the signal sequence from the hGH portion of the protein to produce 
mature hGH. 
BACKGROUND 
Human growth hormone (hGH) is secreted in the human pituitary. In its 
mature form it consists of 191 amino acids, has a molecular weight of 
about 21,500, and thus is more than three times as large as insulin. Until 
the advent of recombinant DNA technology, hGH could be obtained only by 
laborious extraction from a limited source--the pituitary glands of human 
cadavers. The consequent scarcity of the substance has limited its 
application to treatment of hypopituitary dwarfism even though it has been 
proposed to be effective in the treatment of burns, wound healing, 
dystrophy, bone knitting, diffuse gastric bleeding and pseudarthrosis. In 
fact, available estimates are that the amount of hGH available from tissue 
is adequate only to serve about 50 percent of the victims of hypopituitary 
dwarfism. Thus, no hGH is available for other applications. 
Recently, it has been shown that hGH can be produced in a recombinant host 
cell, specifically E. coli in quantities which would be adequate to treat 
hypopituitary dwarfism and the other conditions for which it is effective. 
See, for example, U.S. Pat. No. 4,342,832. While this advance in the art 
promises relief to those who suffer the afflictions for which it offers 
hope of amelioration, for reasons which are set forth below, the hGH 
obtained using the process of U.S. Pat. No. 4,342,832 contains at least a 
substantial amount of hGH to which the amino acid methionine not found in 
native hGH is appended at the N-terminal end of the protein. While there 
is no evidence that this slightly different hGH will, in sensitive 
individuals, cause any important undesirable side reactions it is, 
nevertheless, structurally distinct from "mature" hGH. Hormones which 
differ slightly from those produced by the human body, such as various 
insulins obtained as tissue extracts of cattle and other animals, have 
been successfully used to treat human disease for many years. 
Nevertheless, the advent of recombinant DNA techniques have made it 
possible to obtain insulin of precisely the same amino acid sequence as 
that produced by the body. This has been hailed not only as a great 
scientific advance but a medical one as well since the availability of a 
process for making human insulin promises to reduce the risk of adverse 
side reactions attendant with ingestion of animal insulin to those who 
suffer diabetes. Therefore, notwithstanding the availability of hGH in an 
active form which differs only slightly from that occurring naturally, 
there remains a need to obtain hGH conveniently which, in its amino acid 
content, consists solely of the 191 amino acid sequence of the hGH 
produced by the pituitary. Further, the herein invention discloses the 
production of met-less hGH in commercially practicable amounts. 
The use of recombinant DNA technology to obtain vectors for expressing 
heterologous DNA in a transformed microbial host is now a well established 
science. The first successes in this field were achieved using strains of 
the gram-negative bacterium E. coli such as E. coli K-12, strain 294. 
The use of E. coli as a microbial host for obtaining complex heterologous 
polypeptides has its limitations however. Relatively small polypeptides 
must be obtained as a fusion protein in which the target polypeptide is 
expressed as part of a larger polypeptide in order to protect the small 
protein from degradation by the host cell. For most purposes, the small 
protein produced as a fusion protein must be cleaved in some way from the 
larger molecule to obtain a useful product. 
Large foreign proteins are not degraded by the cell and can be produced 
directly if the gene for their direct expression, including the 
appropriately placed start codon, is linked to a suitable promoter gene, 
such as the well known lac promoter. The signal to begin translation of 
the mRNA coding sequence is the AUG generated from the ATG gene codon 
which also codes for the amino acid methionine (Met). Because prokaryotes 
sometimes do not remove the N-terminal Met from the resulting protein, 
expression of heterologous DNA under control of a bacterial promoter and 
in a bacterial host sometimes results in a protein whose first amino acid 
is methionine. Results to date, for example, with production of hGH in E. 
coli, have shown that the host cell has only a limited ability to cleave 
methionine intracellularly and there is no convenient way to do so 
extracellularly. Accordingly, as noted above, microbially expressed hGH by 
the process of U.S. Pat. No. 4,342,832 leads to a product in which at 
least a substantial portion of the hGH has the appended methionine group 
which, in some circumstances, may cause the protein to be recognized as a 
foreign protein when used in therapeutic applications. 
Many naturally occurring proteins are initially expressed in their normal 
environment with an additional peptide sequence which permits the protein 
to pass through a cellular membrane of the cell in which it is 
manufactured. The additional peptide, which is cleaved in this process, is 
referred to as a "signal" peptide. If a heterologous gene which included 
the gene for a signal sequence were placed under control of a bacterial 
promoter and the bacterium would cleave the signal sequence 
intracellularly, the mature protein without an appended methionine moiety 
could be obtained. However, unless cleaved by the host, the signal 
sequence actually complicates isolation of the mature protein since 
extracellular cleavage is not easily accomplished. 
Efforts to produce "immature" protein, i.e., the protein of interest 
coupled to a signal sequence, in E. coli have suggested that gram-negative 
bacteria such as E. coli do not effectively process this protein to cleave 
the signal sequence, however. A small protein preproinsulin, has been 
shown to be partly processed to remove the signal peptide in E. coli. 
However, no success at all has been obtained with large molecules such as 
fibroblast and leukocyte interferons. In the case of fibroblast 
interferon, no biologically active material was produced (Taniguchi, T. et 
al., Proc. Natl. Acad. Sci. USA 77, 5230-5233 (1980)). In the case of the 
leukocyte interferons, biologically active material was produced but was 
neither transported nor properly processed. 
SUMMARY OF THE INVENTION 
We have found, unexpectedly, that the gene for pre-hGH, i.e., a gene coding 
for the 191 amino acids of the mature protein and the 26 amino acids of 
its signal peptide, is expressed to give pre-hGH in gram negative bacteria 
which is then processed in the cell to cleave the signal peptide from the 
mature protein. (Pre-hGH or pre-protein refers to the desired protein 
containing a signal sequence which, in its native environment effects 
secretion of the desired protein.) As a result, hGH is obtained in its 
mature form, and in an environment in which it is free of other proteins 
associated with its native environment. Thus, using the process of the 
invention it can be obtained free of proteins of human origin, in 
commercially useful amounts, and without the superfluous methionine in 
addition to the amino acid sequence of the naturally occurring protein. 
The present invention also provides replicable vectors for the expression 
of the gene for the immature protein which can be used in both E. coli and 
Pseudomonas and other prokaryotic bacterial species. The invention further 
provides prokaryotic hosts transformed by such vectors. 
An object of the present invention, therefore, is an improved process for 
obtaining hGH by recombinant DNA technology. 
Yet another object is to obtain hGH which is free of appended amino acids 
not found in the natural form. 
The achievement of these and other objects will be apparent from the 
following discussion of presently preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION 
The general approach to the invention is the preparation of an expression 
vector or cloning vehicle which is replicable in the host prokaryote and 
which contains a DNA sequence which codes for expression of immature hGH 
operably connected to a DNA which effects expression. As used herein, 
"prokaryote" refers to cells which do not contain a nucleus and whose 
chromosomal material is thus not separated from the cytoplasm. Prokaryotes 
include, for example, bacteria but do not include such nucleated 
microorganisms as yeast. 
Specifically, plasmids were constructed as expression vectors which could 
be used to transform both E. coli and Pseudomonas strains in order to 
demonstrate the ability of bacterial hosts to effect expression of pre-hGH 
and process it to cleave the signal sequence. 
It has been shown previously that the E. coli plasmid pBR322, which is the 
basic plasmid modified for expression of heterologous DNA in E. coli, can 
be maintained stably in Pseudomonas (Ps.) aerugenosa (a.) if cloned in the 
broad host range, sulfonamide resistant (Su.sup.R), streptomycin resistant 
(Sm.sup.R) plasmid RSF1010, which is also an E. coli plasmid. See Wood et 
al., J. Bacteriol., 14, 1448 (1981) and Sakagouchi, Current Topics in 
Microbiology and Immunology, 96, 31 (1982). Therefore, to obtain plasmids 
which would code for hGH and which could be used to transform both E. coli 
and Ps. strains, we determined to prepare hybrid plasmids of pBR322 and 
pRSF1010 which contained genes for the expression of that protein. 
The construction of a plasmid to code for the expression of immature hGH, 
i.e., for the 191 amino acids of hGH linked to the 26 amino acids of its 
signal sequence, is shown in FIG. 1B. The construction uses a pBR322 
derivative, plasmid phGH207-1 described in de Boer, H. et al. Promoters: 
Structure and Function, eds. Rodriguez, R. and Chamberlin, M. J. (Praeger, 
New York), 462-481 (1982). The phGH207-1, designated 1 in FIG. 1B, was 
partially digested with EcoRI and the largest fragment purified by 
electrophoresis on polyacrylamide gel. This fragment was subjected to 
second strand synthesis with DNA polymerase Klenow fragment and ligated 
with T4 DNA ligase to form a plasmid, phGH207-1*, with a unique EcoRI site 
at the junction of the trp promoter-ribosome binding site and the hGH 
structural gene. The phGH207-1* was partially cleaved with PstI and the 
largest fragment purified by electrophoresis on a polyacrylamide gel. This 
fragment was subjected to second strand synthesis with DNA polymerase 
Klenow fragment and ligated with T4 DNA ligase to form plasmid 
phGH207-1*-APS, designated 2 with a unique PstI site within the hGH 
structural gene. The phGH207-1*-APS was digested to completion with EcoRI 
and PstI and the largest fragment purified by gel electrophoresis using a 
polyacrylamide gel. This fragment contains the trp promoter and the 5' end 
of the gene of mature hGH. 
A second plasmid, phGHcDNA designated 4 in FIG. 1B, was prepared as 
described by Martial et al., Science 205, 602-606 (1979) and treated with 
HpaII to excise a 462 base pair (bp) fragment which codes for most of the 
signal peptide DNA sequence of hGH and the amino terminal portion of 
mature hGH. This fragment was purified by gel electrophoresis using 
polyacrylamide gel. This fragment was digested with PstI to excise a 192 
bp fragment which codes for most of the signal peptide DNA sequence of hGH 
and the amino-terminal portion of mature hGH. This fragment was purified 
by gel electrophoresis using polyacrylamide gel. 
Referring now to FIG. 1A, there is shown the amino acid and mRNA sequences 
for the signal polypeptide of immature hGH with codons AUG for f-Met, the 
signal for initiation of translation in bacteria, which reveals that the 
HpaII site is near the 5' end. To complete the gene for the signal 
sequence and to provide EcoRI and HpaII sites, the two synthetic 
oligonucleotides 2 in FIG. 1B were synthesized by the improved triester 
method of Crea, Proc. Nat'l. Acad. Sci. USA, 75, 5765 (1978). Aliquots of 
5 ug of each of the two synthetic oligonucleotides were phosphorylated 
using T4 polynucleotide kinase and [.gamma.-.sup.32 P]ATP (NEN) as 
described by Goeddel et al, Proc. Nat'l. Acad. Sci. USA, 76, 106 (1979). 
The two molecules were then annealed by mixing these reaction products, 
heating for 5 min. at 90.degree. C. and then cooling to 22.degree. C. 
The fragments phGH207-1 and phGHcDNA and the synthetic oligonucleotides 
were, in a three way ligation using T4 ligase, joined to obtain plasmid 
pPrehGH207-1, designated 5 in FIG. 1B, which was cloned in E. coli K-12, 
strain 294 (E. coli 294), which has been deposited with the American Type 
Culture Collection, ATCC Accession No. 31446, on Oct. 28, 1976. The 
plasmid was isolated from a colony which was Tc.sup.R. The nucloeotide 
sequence of the region comprising the trp promoter and the codons for the 
hGH signal polypeptide and the amino terminal amino acids was confirmed by 
the dideoxy chain termination method. See Messing, J., Crea, R., and 
Seeburg, P. H., Nucleic Acids Res. 9, 309-321 (1981). 
The pPrehGH207-1 and phGH207-1 were digested with XbaI and BamHI. The 
smaller fragment of pPrehGH207-1 was purified by gel electrophoresis and 
contains the entire pre hGH gene. The larger fragment of phGH-207-1 was 
purified by gel electrophoresis and contains the trp promoter. These two 
fragments were mixed and treated with T4 DNA ligase to give plasmid 6, 
designated pPrehGH207-2, which contains the trp promoter and the pre hGH 
gene. The pPrehGH207-2 and pRSF1010 designated 7 were treated with EcoRI 
and joined with T4 ligase to give plasmid 8, designated pRPH-2, which was 
used to transform E. coli 294. The pRPH-2 was obtained from a colony 
exhibiting Tc.sup.R and Sm.sup.R. 
The expression vector pRPH-2 was used to transform Ps.a. strain 2003, 
Chandler et al, Mutat. Res., 23, 15 (1974). Cells transformed with pRPH-2 
were grown overnight in Luria broth (LB) with 50 .mu.g/ml tetracycline at 
37.degree. C. to logarithmic phase. Cell pellets were resuspended in 30 mM 
Tris, pH 8.0, 20.DELTA.sodium dodecyl sulfate (SDS) and sonicated. The 
cell extracts were serially diluted for analysis by radioimmunoassay using 
a Phaedabas hGH PRIST kit (Pharmacia). Substantial levels of hGH were 
found (4.times.10.sup.2 ng/ml/A.sub.550). Cell extracts were 
electrophoresed in 12.5 percent SDS on polyacrylamide gels. hGH was 
visualized by an immunoblotting technique using anti-hGH rabbit anti-serum 
(supplied by Kabi) and .sup.125 I-labeled protein a. The cell extracts 
were shown by autoradiography to contain one major component reactive with 
anti-hGH which has the same electrophoretic mobility as authentic 
pituitary hGH. A minor band of somewhat lesser mobility was also detected 
and is presumably unprocessed pre hGH. 
The major reactive component of the cell extracts was purified to 
homogenity by immunoaffinity chromatography and high performance liquid 
chromatography, and the amino acid sequence of the amino-terminus was 
determined by the Edman degradation method. See Edman et al, European J. 
Biochem., 1, 80 (1967). It was found to be homogeneous and to begin with 
the sequence Phe-Pro-Thr-Ala in perfect correspondence to the sequence of 
pituitary hGH. 
Expression of pre hGH accompanied by proteolytic cleavage to obtain the 
mature hGH using pRPH-2 transformants of E. coli and Ps. putida has also 
been accomplished indicating that the ability of gram negative bacteria to 
successfully process the pre hGH, although unexpected in view of the prior 
failure of E. coli to process interferons possessing their own signal 
peptides, is a general phenomenon. 
In view of the foregoing those skilled in the art can appreciate that 
modifications of the preferred embodiments can be made without departure 
from the spirit of the invention. For example, it is not necessary to use 
a plasmid of pRSF1010 if the recombinant host to be employed is E. coli. 
Thus, successful expression in E. coli can be achieved using, for example, 
pPrehGH207-1 or pPrehGH207-2 of FIGS. 1A and 1B. Accordingly, the present 
invention should be limited only by the lawful scope of the appended 
claims.