Expression of human parathyroid hormone in Escherichia coli

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.     

Expression of human parathyroid hormone in Escherichia coli

Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-beta-galactosidase-hPTH fusion protein under temperature-sensitive control of the lambda phage PR promoter. The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues. Up to 250 mg of pure fusion protein have been obtained from 1-liter E. coli culture by stepwise solubilization with urea. The linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A simple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclase identically to bovine or human PTH.     

High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli

A fully active analog of human parathyroid hormone (hPTH) has been produced by recombinant expression in Escherichia coli. Initially, a nucleotide sequence encoding hPTH(1-34)-Asp-Pro was ligated to a proinsulin gene in the plasmid pUC8, for the eventual expression of a fusion protein of 137 amino acids. Unexpectedly, the proinsulin gene and 340 bp downstream were deleted by an unknown mechanism during transformation of the E. coli. This resulted in a new plasmid encoding a small (72-amino acid) fusion product of hPTH(1-34)-Asp35-Pro36-X, where X is a 36-residue "arbitrary" downstream sequence of pUC8. The fusion product was efficiently expressed and the hPTH analog, [Asp35]hPTH-(1-35), was readily released by acid cleavage, with a yield of 100 mg/L. This analog had an effective concentration for half-maximal adenylyl cyclase stimulation (EC50) in rat osteosarcoma cells of 14 nM, which was identical to that for hPTH-(1-34). In the ovariectomized rat model of osteoporosis, [Asp35]hPTH-(1-35) was fully active as a bone anabolic agent.     

Expression and secretion of human epidermal growth factor by Escherichia coli using enterotoxin signal sequences

Four synthetic genes encoding the signal sequences of Escherichia coli enterotoxins, ST-I, ST-II, LT-A, and LT-B, were incorporated into vectors for expression and secretion of human epidermal growth factor (hEGF) in E. coli. Except for the ST-I signal peptide, these were correctly processed, releasing hEGF from the cytoplasm. The amount of hEGF produced was conveniently monitored with HPLC. The highest level of secretion was obtained with the host carrying the plasmid containing the ST-II signal peptide. The signal peptidase cleaving site in the ST-I signal sequence was determined and found to differ from those previously proposed.     

Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli

Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.     

Periplasmic secretion of human growth hormone by Escherichia coli

The gene coding for human growth hormone (hGH) was fused to the coding sequence for the signal peptide of a secreted Escherichia coli protein. STII heat-stable enterotoxin. This hybrid gene was expressed in E. coli. The signal peptide is properly processed and hGH is secreted in to the periplasmic space. In E. coli, some of the material made is proteolytically clipped or deamidated. The effect of culture conditions on the expression and secretion of hGH was studied and several important parameters were identified, including culture temperature and duration, cultivation pH, K+ levels, plasmid structure, and nutrient supplements. Alteration of culture conditions significantly improves the recovery yield and product quality of human growth hormone.     

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830: pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage p_R promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30°C; the temperature shift to induce production of fusion protein was varied from 36 to 42°C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38°C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/l per hour). By applying a fedbatch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.     

Expression and characterization of a recombinant human parathyroid hormone secreted by Escherichia coli employing the staphylococcal protein A promoter and signal sequence

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids (hPTH(1-84)). Employing the promoter and signal sequence of Staphylococcus aureus-protein A we have expressed hPTH in Escherichia coli. The expressed proteins are excreted to the growth medium, allowing for rapid and easy purification of the desired products. By amino acid sequence analysis and mass spectrometry, we have shown that the major excreted product is correctly processed human identical hPTH(1-84). The purified recombinant hPTH(1-84) stimulates adenylate cyclase activity in rat osteosarcoma cell membranes to exactly the same extent as synthetic parathyroid hormone standards, indicating that the recombinant product has full biological activity.     

Periplasmic production of correctly processed human growth hormone in Escherichia coli: natural and bacterial signal sequences are interchangeable

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 disulfide bonds as authentic hGH.     

Specific degenerate codons enhanced selective expression of human parathyroid hormone in Escherichia coli

Specific degenerate codons in the amino-terminal region of a synthetic human parathyroid hormone (PTH) gene exerted dramatic effects on both products and yield of expression of this 84-amino acid polypeptide in Escherichia coli. With adenine-rich degenerate codons constituting the PTH-(1-5) region, intact PTH has been expressed as the only PTH product at 6.5 mg/liter. In contrast, with guanine-rich degenerate codons, the predominent product was analogue PTH-(8-84). Use of cytosine- or thymine-rich degenerate codons generated only a small amount of immunoreactive product (0.2 mg/l). With the amino terminal region reconstituted with adenine-rich degenerate codons, the mid and carboxyl regions of the synthetic gene were also reconstructed to imitate the E. coli-favored codon degeneracy. Expression yielded the intact PTH at 20 mg/liter. Gel electrophoresis and Western blots, with antibodies specific to the amino or carboxyl terminus of PTH, indicated only a single PTH-related polypeptide, with the same mobility as a synthetic intact PTH sample. Amino acid sequencing, composition analysis, mass spectrometry, and the adenylate cyclase bioassays confirmed the purified product as the processed intact PTH.     

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.     

Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli

Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.     

Improved solubilization of recombinant human growth hormone inclusion body produced in Escherichia coli

Recombinant human growth hormone (r-hGH) overexpressed in Escherichia coli forms inactive and insoluble aggregates as inclusion bodies in the cytoplasm. The efficient solubilization of inclusion bodies is critical for cost-effective production. Contrary to a previous report, in our production system, the solubilization method by alkaline treatment including 2 M urea was ineffective. Hence various buffers containing different concentrations of urea or guanidine hydrochloride (GnHCl) at neutral and alkaline pH were attempted. Efficient solubilization (about 90%) was observed in 100 mM Tris buffer, pH 8.0, with more than 4 M GnHCl, and at pH 12.5 with more than 2 M GnHCl, but not with about 8 M of urea. The r-hGH solubilized at pH 12.5 containing 2 M GnHCl was refolded by simple dilution and purified by DEAE Sepharose anion-exchange chromatography. The biological activity of the resulting r-hGH was comparable with commercially available r-hGH in in vitro cell proliferation assay using the hGH-dependent cell line.     

A signal transducer for aerotaxis in Escherichia coli.

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.     

Full-length chicken parathyroid hormone. Biosynthesis in Escherichia coli and analysis of biologic activity.

Chicken parathyroid hormone (cPTH) has been reported to stimulate adrenal steroidogenesis and to have unusual potency on traditional PTH target tissues. To evaluate these properties, chicken PTH-(1-88) has been expressed in Escherichia coli using a plasmid encoding a fusion protein which links together growth hormone, a factor Xa recognition site, and chicken PTH-(1-88). The growth hormone-cPTH fusion protein required the presence of 0.02% sodium dodecyl sulfate to remain in solution and be cleaved by factor Xa. The high performance liquid chromatography-purified recombinant cPTH-(1-88) and chemically synthesized cPTH-(1-34) had similar potency in rat osteosarcoma (ROS 17/2.8) cells, opossum kidney (OK) cells, and dispersed primary chicken kidney cells. The biologic potencies of cPTH-(1-34) and cPTH-(1-88) in radioreceptor binding and cAMP generation in both bone- and kidney-derived cell lines were less than those of human (h)PTH-(1-34). In dispersed chicken kidney cells, cAMP production by cPTH-(1-34) and cPTH-(1-88) was similar to that stimulated by human PTH-(1-34). No stimulation of steroidogenesis could be detected when recombinant chicken PTH-(1-88) was added to dispersed chicken adrenal cells. The biologic activity of recombinant chicken PTH-(1-88) purified from E. coli was comparable with that of chicken PTH-(1-88) expressed by mammalian COS cells. Thus, the full-length chicken PTH did not exhibit enhanced potency, when compared with human PTH in ROS 17/2.8, OK cell lines, and dispersed chicken kidney cells and did not demonstrate the novel steroidogenic action previously reported in adrenal cells. The successful production of chicken PTH-(1-88) will enhance our understanding of the structure-activity relationships for PTH, particularly the sequence-dependent metabolism of the hormone.     

In vitro processing of pro-subtilisin produced in Escherichia coli

In a previous paper (Ikemura, H., Takagi, H., and Inouye, M. (1987) J. Biol. Chem. 262, 7859-7864), we demonstrated that the pro-sequence consisting of 77 amino acid residues at the amino terminus of subtilisin is essential for the production of active subtilisin. When the aggregates of pro-subtilisin produced in Escherichia coli were solubilized in 6 M guanidine hydrochloride and dialyzed against 200 mM sodium phosphate buffer (pH 7.1 or 6.2), pro-subtilisin was efficiently processed to active subtilisin. When more than 14 residues were removed from the amino terminus of the pro-sequence, active subtilisin was no longer produced as in the in vivo experiments. Similarly, active subtilisin would not renature under the same conditions once solubilized in guanidine hydrochloride. When the aspartic acid residue at the active site (Asp32) was altered to asparagine, processing of mutant pro-subtilisin was not observed even in the presence of wild-type pro-subtilisin. Inhibitors such as phenylmethanesulfonyl fluoride or Streptomyces subtilisin inhibitor did not block the processing of wild-type pro-subtilisin. These facts indicate that processing or pro-subtilisin is carried out by an intramolecular, self-processing mechanism. When the sample was dialyzed against 20 mM sodium phosphate (pH 6.2), no active subtilisin was found, suggesting that the highly charged nature of the pro-sequence plays an important role in the process of refolding of denatured pro-subtilisin.     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli

Human parathyroid hormone (hPTH) is a promising agent in the treatment of osteoporosis. The intact recombinant human parathyroid hormone [rhPTH(1-84)] was prepared in a large scale from Escherichia coli using a soluble fusion protein strategy. With degenerate codons, gene of hPTH(1-84) was synthesized, ligated with pET32a(+) vector, and then expressed in E. coli BL21(DE3) cells. The soluble fusion protein His(6)-thioredoxin-hPTH(1-84) was harvested after purification by immobilized metal affinity chromatography (IMAC). Following enterokinase cleavage, ion-exchange-chromatography (IEC) and size-exclusive-chromatography (SEC) were used, and finally, over 300mg/l intact hPTH(1-84) with high purity up to 99% was obtained. The purified rhPTH(1-84) was confirmed by mass spectrometry and N-terminal/C-terminal amino-acid sequence analysis. Additionally, this product stimulated adenylate cyclase in Rat Osteosarcoma Cell UMR-106 at the same extent as hPTH standards, indicating that the purified rhPTH(1-84) has full biological activity. The efficient procedure for expression and purification of rhPTH(1-84) may be useful for the mass production of this important protein.