Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.     

Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis

A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction. This plasmid was transformed into Escherichia coli strain M15 and effectively expressed. The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column. The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system. The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride. Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column. These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein. Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.     

GCN2 Protein Kinase Is Required to Activate Amino Acid Deprivation Responses in Mice Treated with the Anti-cancer Agent l-Asparaginase

Asparaginase depletes circulating asparagine and glutamine, activating amino acid deprivation responses (AADR) such as phosphorylation of eukaryotic initiation factor 2 (p-eIF2) leading to increased mRNA levels of asparagine synthetase and CCAAT/enhancer-binding protein β homologous protein (CHOP) and decreased mammalian target of rapamycin complex 1 (mTORC1) signaling. The objectives of this study were to assess the role of the eIF2 kinases and protein kinase R-like endoplasmic reticulum resident kinase (PERK) in controlling AADR to asparaginase and to compare the effects of asparaginase on mTORC1 to that of rapamycin. In experiment 1, asparaginase increased hepatic p-eIF2 in wild-type mice and mice with a liver-specific PERK deletion but not in GCN2 null mice nor in GCN2-PERK double null livers. In experiment 2, wild-type and GCN2 null mice were treated with asparaginase (3 IU per g of body weight), rapamycin (2 mg per kg of body weight), or both. In wild-type mice, asparaginase but not rapamycin increased p-eIF2, p-ERK1/2, p-Akt, and mRNA levels of asparagine synthetase and CHOP in liver. Asparaginase and rapamycin each inhibited mTORC1 signaling in liver and pancreas but maximally together. In GCN2 null livers, all responses to asparaginase were precluded except CHOP mRNA expression, which remained partially elevated. Interestingly, rapamycin blocked CHOP induction by asparaginase in both wild-type and GCN2 null livers. These results indicate that GCN2 is required for activation of AADR to asparaginase in liver. Rapamycin modifies the hepatic AADR to asparaginase by preventing CHOP induction while maximizing inhibition of mTORC1.     

Effect of medium composition on the growth and asparaginase production of Vibrio succinogenes.

Asparaginase synthesis by Vibrio succinogenes is induced by ammonium ions. Synthesis occurs throughout exponential phase, and in early stationary phase asparaginase accounts for about 5% of the total soluble protein. The organism grows best when fumarate is provided as the terminal electron acceptor of the formate-oxidizing cytochrome system. Yeast extract or enzyme-hydrolyzed proteins are effective nutrient sources. In an ammonium formate-sodium fumarate medium, where maximum growth and asparaginase synthesis occurs, the total enzyme yield (international units per liter of culture) is about one-tenth that obtainable with a good asparaginase-producing strain of Escherichia coli. The energetic inefficiency of V. succinogenes appears to cause a low yield of cells and therefore low total enzyme yield. However, the levels of asparaginase accumulated within cells raise questions about the organism's protein synthesizing system.     

Equilibrium denaturation of human growth hormone and its cysteine-modified forms

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this.     

Refolding of porcine growth hormone from inclusion bodies of Escherichia coli

Escherichia. coli cells expressing porcine growth hormone were grown in a batch fermentation process. The expression level was estimated to be nearly 40% of the total cellular protein after 2–3 h of induction with 1?mM isopropyl β-d-thiogalactoside. Porcine growth hormone expressed as inclusion bodies was solubilized in 8 M urea. Refolding conditions following a dilution protocol in the presence of β-mercaptoethanol or using a glutathione pair were tested. Reverse phase-HPLC was applied to distinguish oxidized, misfolded and reduced forms of the hormone. A ratio of reduced to oxidized glutathione equal to 2/1 was chosen to avoid the formation of misfolded forms at high protein concentration.     

L-asparaginase from developing seeds of Lupinus arboreus.

Asparaginase (EC 3.5.1.1) activity reached a maximum 40 days post anthesis in developing seeds of Lupinus arboreus and this correlated with the appearance of other ammonia assimilatory enzymes. Asparaginase, purified from these developing seeds, was resolved into three isoforms, designated asparaginases A, B and C. A major protein species in asparaginase A preparations co-focussed with enzyme activity on an isoelectric focussing gel. When analysed by SDS-PAGE, asparaginase isoforms A and B each yielded several polypeptides with M(r)s in the 14,000 to 19,000 ranged. These peptides are fragmentation products of an M(r) 36,000 asparaginase subunit. Polyclonal antibodies raised against asparaginase isoforms A and B precipitated asparaginase activity from a partially purified L. arboreus seed extract. Immunoaffinity chromatography recovered polypeptides with M(r)s between 14,000 and 19,000. Partial protein sequences were obtained for these asparaginase polypeptides.     

Aggregation of ALS mutant superoxide dismutase expressed in Escherichia coli

Although large amounts of wild-type human Cu,Zn superoxide dismutase (SOD) are easily expressed in Escherichia coli, the amyotrophic lateral sclerosis-associated mutants have a strong propensity to aggregate into inclusion bodies. The alanine to valine mutation at the fourth codon (A4V) is responsible for a rapidly progressive disease course and is particularly prone to aggregation when expressed in E. coli. We found that A4V SOD remained soluble when expressed at 18 degrees C, but >95% A4V SOD aggregated in inclusion bodies when expressed at 23 degrees C or above. The SOD aggregates dissolved with 4 M urea, suggesting that intermolecular hydrophobic interactions were predominantly responsible for making SOD insoluble. Many of the urea-solubilized subunits were cross-linked via disulfide bridges. Fully active mutant SOD could be produced by dialyzing urea away in the presence of beta-mercaptoethanol and subsequently adding copper plus zinc, providing a fast procedure for purifying hundreds of milligrams of protein. Extensive rinsing removed most contaminating E. coli proteins from A4V SOD inclusion bodies except for a 37 kDa protein identified as outer membrane protein F using MALDI ToF/ToF mass spectrometry. Our results indicate that metal-deficient ALS-mutant SOD folds into stable apo conformation able to rebind metals. At high protein concentrations, SOD forms aggregates through hydrophobic interactions between subunits that seem to act as a kinetic snare to entrap additional proteins.     

Synthesis and expression of a human growth hormone (somatotropin) gene mutated to change cysteine-165 to alanine

We have mutated a synthetic human growth hormone (hGH) gene specifically at the codon for Cys-165 to a codon for Ala by replacement of synthetic deoxyoligonucleotides corresponding to this site. This modification prevented the formation of a disulfide bond between Cys-53 and Cys-165 in the hGH molecule. This mutated protein, [Ala165]hGH was expressed at the same level as the intact hGH, 4 X 10(5) molecules per cell under the control of the tryptophan promoter in Escherichia coli, and retained similar immunological activity to intact hGH. The limited digestion pattern of the mutated protein with human plasmin suggests that the tertiary structure of [Ala165]hGH resembles to that of the intact hGH molecule. [Ala165]hGH revealed full biological activity as examined by the body weight increase of hypophysectomized rats.     

HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。     

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.     

重组戊型肝炎病毒衣壳蛋白工程菌的高密度培养

在10L发酵罐中对戊型肝炎病毒衣壳蛋白在重组大肠杆菌中表达发酵工艺进行了研究,用分批培养方法探讨了不同培养基、培养基中磷酸盐浓度和Mg^2＋浓度等因素对菌体生长与重组蛋白表达的影响;用分批补料培养研究了不同的补料工艺对菌体生长与重组蛋白表达的影响,同时对重组菌诱导时期、诱导持续时间以及不同诱导温度表达包含体在尿素溶液中的溶解性进行了研究。结果表明,在优化后的培养基中,磷酸盐浓度、Mg^2＋浓度分别为80mmol/L 与20mmol/L时菌体生长与表达效果较好;分批补料培养中,37℃培养9h菌体达到对数期中期(约45OD600)为适宜诱导时期,加入终浓度为10mmol/L IPTG后诱导5h,OD600达到80以上,重组蛋白表达量达到29.74％,为最适收获菌体时间;37℃表达的包含体80％以上溶解在4mol/L的尿素溶液中,最终浓度达到14mg/mL; 10L发酵罐中确定的发酵工艺参数在30L发酵罐中进行了放大培养,10L发酵罐中确定的发酵工艺参数在30L发酵罐上具有可放大性与重复性, 可以应用于工业生产。     

Role of glucose utilization in the restoration of hypophysectomy-induced hepatic cytochrome P450 2E1 by growth hormone in rats

Growth hormone and insulin are the primary determinants for cytochrome P450 2E1 (CYP2E1) expression. The role of glucose on the induction of CYP2E1 by hypophysectomy and on the restorative effect by growth hormone was investigated in the rat liver. Western and Northern blot analyses revealed that hypophysectomy induced CYP2E1 by 5-fold at 1-4 weeks, relative to control, with a concomitant increase in CYP2E1 mRNA. Hypophysectomized rats (HXR) showed a 20% reduction in the plasma glucose level. Hypophysectomy-induced increase in the CYP2E1 mRNA was completely abolished by glucose feeding in drinking water (10%) for 7 days. Treatment of HXR with hGH (2 I.U./kg, twice a day, for 7 days) inhibited the increases in CYP2E1 protein and mRNA levels with restoration of the plasma glucose level. In contrast to the effect of human growth hormone (hGH) on CYP2E1 in HXR with free access to foods, CYP2E1 expression failed to be restored by hGH in starving HXR. However, glucose feeding of starving HXR abolished the induction of CYP2E1. Effects of hypophysectomy and hGH treatment were studied in streptozotocin-induced diabetic rats. Insulin, but not hGH, prevented an increase in CYP2E1 mRNA in diabetic rats. The hepatic CYP2E1 induction in hypophysectomized diabetic rats was inhibited by hGH treatment, indicating that the hGH effect on CYP2E1 expression did not involve insulin production. These results provide evidence that the induction of hepatic CYP2E1 by hypophysectomy may result from reduced glucose utilization, and that the effect of hGH on CYP2E1 expression may be mediated with enhanced glucose utilization, but not with insulin production.     

Refolding and structural characteristic of TRAIL/Apo2L inclusion bodies from different specific growth rates of recombinant Escherichia coli

TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) was produced mainly as inclusion bodies (IBs) by recombinant Escherichia coli with a temperature-inducible expression system. The yield of TRAIL type 2 IBs at higher preinduction specific growth rate (mu = 0.15 h-1) was higher than that of TRAIL type 1 IBs at lower preinduction specific growth rate (mu = 0.05 h-1). With the same optimized refolding protocols, two types of IBs exhibited different refolding features. Refolded type 1 IBs had higher recovery of more than 80% compared with type 2 IBs (57-63%). By the measurements of fluorescence and CD spectroscopy, type 1 TRAIL IBs dissolved by urea appeared to be a closer secondary structure to the native TRAIL than type 2. Furthermore, with trypsin treatment, the striking decrease in stability of type 1 IBs against protease digestion cannot be attributed to their small size particles observed by scanning electron microscope and probably depend on different protein structure properties between the two IBs. Different properties of inclusion bodies were mainly influenced by different physiological states of the cells just prior to the induction.     

Isolation of four isomers of the 20,000 dalton variant of human pituitary growth hormone

A simple procedure has been developed which for the first time describes the isolation of isomers of the 20,000 dalton variant of human growth hormone (20K hGH). From a human pituitary hormone concentrate different hGH dimers (covalently and noncovalently linked) were enriched by chromatography on SP-Sephadex C-50, DEAE-Sepharose CL-6B and Sephadex G-100. Noncovalently-linked dimers were split by 6 M urea into 20K hGH and 22K hGH monomers. A complete group-separation of 20K hGH and 22K hGH monomers was achieved by chromatography on DEAE-Sepharose CL-6B at neutral pH. The 20K hGH monomer was resolved into four isomers either by preparative isoelectric focusing or by zone electrophoresis in agarose suspension at alkaline pH. The three latter techniques were all used in the presence of 6 M urea. Radioimmunoassay and radioreceptorassay indicated that the isomers obtained were true components of human growth hormone.     

Expression, Purification, and Encephalitogenicity of Recombinant Human Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.     

Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen

Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties. For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors. We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp). In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex. As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold. We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL. By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL. Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors. Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events.     

Substitution of serine for non-disulphide-bond-forming cysteine in grass carp (Ctenopharygodon idellus) growth hormone improves in vitro oxidative renaturation

Native grass carp (Ctenopharygodon idellus) growth hormone, has 5 cysteine amino acid residues, forms two disulphide bridges in its mature form. Recombinant grass carp growth hormone, when over-expressed in E. coli, forms inclusion bodies. In vitro oxidative renaturation of guanidine-hydrochloride dissolved recombinant grass carp growth hormone was achieved by sequential dilution and stepwise dialysis at pH 8.5. The redox potential of the refolding cocktail was maintained by glutathione disulphide/glutathione couple. The oxidative refolded protein is heterogeneous, and contains multimers, oligomers and monomers. The presence of non-disulphide-bond-forming cysteine in recombinant grass carp growth hormone enhances intermolecular disulphide bond formation and also nonnative intramolecular disulphide bond formation during protein folding. The non-disulphide-bond-forming cysteine was converted to serine by PCR-mediated site-directed mutagenesis. The resulting 4-cysteine grass carp growth hormone has improved in vitro oxidative refolding properties when studied by gel filtration and reverse phase chromatography. The refolded 4-cysteine form has less hydrophobic aggregate and has only one monomeric isoform. Both refolded 4-cysteine and 5-cysteine forms are active in radioreceptor binding assay.