Genetic engineering of peptide hormones

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.     

Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities

Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.     

Single-step purification of recombinant human growth hormone (hGH) directly from bacterial osmotic shock fluids, for the purpose of (125)I-hGH preparation

A good quality tracer, to be used in the radioimmunoassay of human growth hormone, was prepared by applying the chloramine-T iodination technique to the recombinant product obtained after a single-step high-performance size-exclusion chromatography purification of a bacterial osmotic shock fluid. The labeling reaction presented a yield of about 65% and the purified tracer exhibited an antibody binding of approximately 50% (NIDDK reference antiserum diluted 1:600,000). These values are very similar to those obtained by radioiodinating highly purified clinical-grade recombinant human growth hormone obtained from the same periplasmic extract after the regular six-step purification process. Both tracers provided the same accuracy, when evaluated with the use of commercial-quality control samples in a classical radioimmunoassay methodology, their stability being practically identical: about 18% decrease in antibody binding after 2 months of storage at -20 degrees C. The novel approach permits the utilization of transformed Escherichia coli strains as a source of freshly prepared, radioiodination-grade recombinant proteins, capable of providing better reproducibility and reagent continuity.     

Heterologous expression and characterization of Indian Sahiwal cattle (Bos indicus) alpha inhibin

Inhibin is a non-steroidal glycoprotein hormone of gonadal origin with major action as negative feedback control of the production of FSH by the anterior pituitary gland. The physiological role of inhibin has led to the development of inhibin immunogens for fertility enhancement in farm animals. It is envisaged that a reduction of endogenous inhibin secretion would increase FSH concentrations and thus offers a potential for increasing the number of ovulatory follicles in the ovary. The present work was carried out to produce recombinant bovine (Indian Sahiwal Cattle; Bos indicus) alpha inhibin (bINH-α) in E. coli by optimizing its expression and purification in biologically active form and to study its immunological characterization. A bacterial protein expression vector system based on the phage T(5) promoter was used. The bINH-α encoding gene was successfully cloned and expressed in E. coli and the purified recombinant bINH-α was characterized. Recombinant bINH-α (25?μg?mL(-1)) immunized guinea pigs had a significant increase in litter size compared to the control group. These results indicate a role for recombinant bINH-α as a fecundity vaccine to enhance the ovulation rate and litter size in animals.     

Biosynthesis and secretion of bovine growth hormone in Escherichia coli under the control of the secretory vector containing a promoter and signal region of the alkaline phosphatase gene

A recombinant plasmid carrying a bovine growth hormone gene fused with the regulatory and signal regions of the alkaline phosphatase gene of E. coli was constructed. The bovine growth hormone gene expression as well as protein partial processing and secretion into the periplasm have been shown to take place under phosphate starvation, i.e. conditions of alkaline phosphatase derepression.     

Immunoassay of serum polypeptide hormones by using 125I-labelled anti(-immunoglobulin G) antibodies.

1. A technique for indirectly labelling antibodies to polypeptide hormones, by combining them with radioactively labelled anti-(immunoglobulin G) is described. (a) 125I-labelled anti-(rabbit immunoglobulin G) and anti-(guinea-pig immunoglobulin G) antibodies with high specific radioactivity were prepared after purification of the antibodies on immunoadsorbents containing the respective antigens. (b) Rabbit immunoglobulin G antibodies to human growth hormone, porcine glucagon and guinea-pig immunoglobulin G antibodies to bovine insulin and bovine parathyroid hormone were combined with immunoadsorbents containing the respective polypeptide hormone antigen. (c) The immunoglobulin G antibodies to the polypeptide hormones were reacted with 125-I-labelled anti-(immunoglobulin G) antibodies directed against the appropriate species of immunoglobulin G,and the anti-hormone antibodies were combined with the hormone-containing immunoadsorbent. (d) 125I-labelled anti-(immunoglobulin G) antibodies and anti-hormone antibodies were simultaneously eluted from the hormone-containing immunoadsorbent by dilute HCl, pH 2.0. After elution the anti-(immunoglobulin G) antibodies and antihormone antibodies were allowed to recombine at pH 8.0 and 4 degrees C. 2. The resultant immunoglobulin G-anti-immunoglobulin G complex was used in immunoradiometric (labelled antibody) and two-site assays of the respective polypeptide hormone. 3. By using these immunoassays, concentrations down to 90pg of human growth hormone/ml, 100 pg of bovine insulin/ml, 80 pg of bovine parathyroid hormone/ml and 150 pg of glucagon/ml were readily detected. Assays of human plasma for growth hormone and insulin by these methods showed good agreement with results obtained by using a directly 125I-labelled anti-hormone antibody in an immunoradiometric assay of human growth hormone or by radioimmunoassay of human insulin. 4. The method described allows immunoradiometric or two-site assays to be performed starting with as little as 450 ng of polypeptide hormone-antibody protein. An additional advantage of the method is that a single iodination of the readily available antibodies to immunoglobulin G allows the establishemnt of several polypeptide hormone assays     

High-level production and purification of Escherichia coli N-acetylneuraminic acid aldolase (EC 4.1.3.3).

The Escherichia coli gene which encodes N-acetylneuraminic acid aldolase was isolated by the polymerase chain reaction, cloned into the inducible expression vector pTTQ18, and overexpressed in E. coli. The high yield of aldolase was achieved through both optimum growth of cells and efficient expression of the aldolase gene (20-30% soluble cellular protein). The recombinant enzyme was purified to homogeneity with an activity of 1.2-2.2 U/mg, which compared favorably with that of commercial preparations of E. coli aldolase (1.1 U/mg) and Clostridium perfringens aldolase (0.4 U/mg). The cloning strategy, fermentation conditions, purification protocol, and activity assay are described.     

Bovine interleukin-1 expression by cultured mammary epithelial cells (MAC-T) and its involvement in the release of MAC-T derived interleukin-8

MAC-T cells, an established bovine mammary epithelial cell line, were utilized to investigate both expression of interleukin-1 (IL-1) mRNA and secretion of IL-1 after Escherichia coli lipopolysaccharide (E. coli LPS) stimulation. In addition, recombinant human IL-1beta, recombinant human IL-1 receptor antagonist (IL-1ra) and a neutralizing goat antibody against type I human IL-1 receptor were used to study the involvement of IL-1 in the release of IL-8. The expression of MAC-T derived IL-1alpha mRNA was correlated to production of IL-1alpha protein as measured by an IL-1alpha sandwich ELISA. Secretion of IL-1alpha was dose- and time-dependent, with a maximal level of 600 pg/ml detectable upon 2-h stimulation with 20 microg of LPS per ml. IL-1ra and the neutralizing antibody significantly blocked the ability of IL-1beta to stimulate secretion of IL-8 by MAC-T cells. During this study, we have demonstrated that MAC-T cells secrete IL-1 in response to LPS stimulation and IL-1 is an important mediator for the release of the bovine IL-8 by MAC-T cells. These results further indicate the potential importance of mammary epithelial cells as a source of immunoregulation in the mammary gland via cytokine elaboration.     

Expression of synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) in Escherichia coli

Synthetic genes encoding bovine and human basic fibroblast growth factors (bFGFs) were assembled and cloned using established Escherichia coli expression plasmids. Transformed E. coli cells were able to synthesize either a fusion protein, comprising the first seven amino acids of beta-galactosidase, a linker fragment and bovine FGF, or genomic human bFGF. The two growth factors were purified from E. coli lysates by cation exchange and heparin-Sepharose affinity chromatography. The purified recombinant proteins were biologically active as monitored by their mitogenic activity for bovine aortic endothelial cells and their angiogenic capacity in the rabbit cornea.     

rhGM—CSF／LIF融合蛋白基因的克隆及表达

利用基因重组技术,人工构建了一个编码五肽Ｇ－Ｓ－Ｇ－Ｇ－Ｓ的基因接头,将ＧＭ－ＣＳＦ和ＬＩＦ的ｃＤＮＡ相连而构成融合基因,将融合基因载入原核表达载体ｐＢＶ２２０后转化大肠杆菌,经热诱导后进行Ｗｅｓｔｅｒｎ印迹反应鉴定证实获得ｒｈＧＭ－ＣＳＦ／ＬＩＦ融合蛋白（简称ｒｈｇＭ－ＬＩＦ）活性测定表明重组的融合蛋白具有两因子双重活性。     

Characterization of Xenopus cytosolic thyroid-hormone-binding protein (xCTBP) with aldehyde dehydrogenase activity

Multiple cytosolic thyroid-hormone-binding proteins (CTBPs) with varying characteristics, depending on the species and tissue, have been reported. We first purified a 59-kDa CTBP from Xenopus liver (xCTBP), and found that it is responsible for major [125I]T(3)-binding activity in Xenopus liver cytosol. Amino acid sequencing of internal peptide fragments derived from xCTBP demonstrated high identity to the corresponding sequence of mammalian aldehyde dehydrogenases 1 (ALDH1). To confirm whether or not xCTBP is identical to xALDH1, we isolated cDNAs encoding xALDH1 from an adult Xenopus hepatic cDNA library. The amino acid sequences deduced from the two isolated xALDH1 cDNAs were very similar to those of mammalian ALDH1 enzymes. The recombinant xALDH1 protein exhibited both T(3)-binding activity and ALDH activity converting retinal to retinoic acid (RA), which were similar to those of xCTBP purified from liver cytosol. The T(3)-binding activity was inhibited by NAD, while the ALDH activity was inhibited by thyroid hormones. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular concentration of free T(3). Communications between thyroid hormone and retinoid pathways are discussed.     

Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.     

Use of modified BL21(DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage

We previously cloned a panel of peanut allergens by phage display technology. Examination of the codons used in these sequences indicated that most of the cDNAs contain an excess of the least used codons in Escherichia coli, namely AGG/AGA, that correspond to a minor tRNA, the product of the dnaY gene. To achieve high-level expression of the peanut allergens, the cDNAs were subcloned into an expression vector of the pET series (Novagen) in order to produce (His)(10)-tagged fusion proteins in conventional E. coli BL21(DE3) cells. The peanut allergens Ara h 1, Ara h 2, and Ara h 6 with an AGG/AGA codon content of 8-10% were only marginally expressed, whereas the peanut profilin Ara h 5, with an AGG/AGA codon content of only 0.8%, was efficiently expressed in these cells. Hence, by using modified BL21(DE3) E. coli cells, namely BL21-CodonPlus(DE3)-RIL cells (Stratagene) with extra copies of E. coli argU, ileY, and leuW tRNA genes, it was possible to attain high-level expression of the proteins affected by rare codon usage. IPTG-induced expression of several recombinant peanut allergens, such as Ara h 1, Ara h 2, and Ara h 6, was greatly increased in these special cells compared to the expression yield achieved by conventional E. coli hosts. The purification of the soluble and the insoluble fraction of Ara h 2 was performed by metal-affinity chromatography and yielded a total of about 30 mg (His)(10)-tagged recombinant protein per liter of culture of transformed BL21(DE3)CodonPlus-RIL cells. This is over 100 times more than achieved by production of Ara h 2 in conventional BL21(DE3) cells.     

The kinetics of bovine growth hormone folding are consistent with a framework model

The framework model of protein folding requires the hydrogen-bonded secondary structure to be formed early in folding (i.e. the formation of secondary structure precedes the tertiary structure) (Kim, P. S., and Baldwin, R. L. (1982) Annu. Rev. Biochem. 51, 459-489). To test the framework model directly the kinetics of bovine growth hormone (bGH) folding were compared utilizing two methods of detection, one that measures the secondary structure (far ultraviolet circular dichroism) and another that measures the tertiary structure (near ultraviolet absorbance). The results demonstrate that, under identical folding conditions, the kinetics observed by far ultraviolet circular dichroism are faster than those observed by ultraviolet absorption. The faster kinetics observed by circular dichroism indicate the existence of a helix-containing intermediate which is consistent with the framework model. The effect of protein concentration and denaturant concentration on the kinetics of refolding were studied. The rate of refolding measured by absorbance and circular dichroism was dependent on protein concentration. The protein concentration dependence on refolding is due to the transient formation of an associated intermediate. The concentration dependence of folding is taken as evidence that folding is a sequential process with partially folded monomers responsible for the observed association effect. At dilute protein concentrations the refolding can be studied independent of the association phenomena. The growth hormones utilized in this study were derived from Escherichia coli through recombinant DNA technology and from bovine pituitaries. The pituitary-derived bGH has been shown to be heterogeneous at the NH2 terminus (Lorenson, M. F., and Ellis, S. (1975) Endocrinology 96, 833-838), whereas the recombinant DNA-derived bGH contains a single NH2 terminus. No differences in the folding kinetics between the recombinant DNA and pituitary derived-bGH were observed. It is concluded that the heterogeneity of the NH2 terminus of growth hormone obtained from bovine pituitaries does not affect the observed in vitro folding kinetics.     

Expression and purification of recombinant poly(A)-specific ribonuclease (PARN)

PARN is a poly(A)-specific ribonuclease that degrades the poly(A) tail of mRNA. We have established conditions for expressing soluble recombinant human PARN. We investigated different Escherichia coli strains, expression vectors, media and growth conditions. We found that PARN expressed from pET33 in BL21(DE3) grown in TB and induced at OD595 approximately 1 with 1 mM IPTG yielded mg amounts of soluble PARN per litre culture. Further, a purification protocol was established to purify PARN. We use His-tag affinity chromatography, HiTrap Q HP ion exchange chromatography and 7-Me-GTP-Sepharose affinity chromatography. This purification procedure render a 90-95% pure PARN. Purified recombinant PARN has enzymatic activity and will be used for further mechanistic and structural studies.     

Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone.

A process for maximizing the volumetric productivity of recombinant ovine growth hormone (r-oGH) expressed in Escherichia coli during high cell density fermentation process has been devised. Kinetics of r-oGH expression as inclusion bodies and its effect on specific growth rates of E. coli cells were monitored during batch fermentation process. It was observed that during r-oGH expression in E. coli, the specific growth rate of the culture became an intrinsic property of the cells which reduced in a programmed manner upon induction. Nutrient feeding during protein expression phase of the fed-batch process was designed according to the reduction in specific growth rate of the culture. By feeding yeast extract along with glucose during fed-batch operation, high cell growth with very little accumulation of acetic acid was observed. Use of yeast extract helped in maintaining high specific cellular protein yield which resulted in high volumetric productivity of r-oGH. In 16 h of fed-batch fermentation, 3.2 g l-1 of r-oGH were produced at a cell OD of 124. This is the highest concentration of r-oGH reported to date using E. coli expression system. The volumetric productivity of r-oGH was 0.2 g l-1 h-1, which is also the highest value reported for any therapeutic protein using IPTG inducible expression system in a single stage fed-batch process.     

Bacterial expression and purification of recombinant bovine Fab fragments.

We have previously described a recombinant phagemid expression vector, pComBov, designed for the production of native sequence bovine monoclonal antibodies (mAb) generated by antibody phage display. Bovine mAb Fab fragments isolated from libraries constructed using pComBov in Escherichia coli strain XL1-Blue, which is routinely used for antibodies expressed on the surface of phage, were expressed at very low yields. Therefore, a study was undertaken to determine optimal growth conditions for maximal expression of bovine Fab fragments in E. coli. By varying the E. coli strain, and the temperature and length of the culture growth, we were able to substantially increase the yield of soluble Fab fragments. A high yield of Fab fragments was found in the culture growth medium, which enabled us to devise a rapid and simple single-step method for the purification of native (nondenatured) Fabs based on immobilized metal affinity chromatography against a six-histidine amino acid carboxyl-terminal extension of the heavy-chain constant region. Using these methods we were able to express and purify antigen-specific bovine Fab fragments from E. coli.     

Isolation, characterization, and distribution of two cDNAs encoding for growth hormone receptor in rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) plays important roles in a vast array of physiological processes, including growth, metabolism, and reproduction. In this study, cDNAs for two unique growth hormone receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 2920 bp and the other of 2820 bp, share 87.2% identity in nucleotide sequence and 85.5% identity in deduced amino acid sequence and presumably arose through gene duplication. The cDNAs encode for putative 593- and 594-amino acid growth hormone receptors (designated GHR1 and GHR2, respectively), each containing a single transmembrane domain and other motifs characteristic of the receptor family. Both GHR1 and GHR2 mRNAs were present in all tissues examined. Trout GHR mRNAs are differentially expressed, both in terms of abundance among tissues and in terms of abundance within selected tissues. GHR1 was more abundant than GHR2 in the brain, whereas GHR2 was more abundant than GHR1 in pancreas and spleen. These findings expand our understanding of the evolution of the GH receptor family and suggest that independent mechanisms serve to regulate the tissue-specific expression of GHR mRNAs.