Optimization of Inclusion Body Solubilization and Renaturation of Recombinant Human Growth Hormone from Escherichia coli (original) (raw)
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Biotechnology Progress, 2010
We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010
2008
Ovine growth hormone was expressed in Escherichia coli in the form of inclusion bodies using the pQE-30 expression vector. In a simple fed-batch fermentation, 800 mg/L of recombinant ovine growth hormone (r-oGH) was produced at a cell concentration of 12 g dry cell weight/L. Inclusion bodies were isolated from cells with >95% purity by extensive washing using detergent, and the r-oGH from the purified inclusion bodies was solubilized in 2 M Tris-HCl buffer at pH 12 containing 2 M urea. The r-oGH solubilized in the above conditions exhibited considerable secondary structure as determined by circular dichroism spectra and was immunologically active. Solubilization of the inclusion body protein with retention of native-like secondary structure gave higher yields during refolding. To suppress protein aggregation, refolding was carried out in gel filtration column. Refolding, buffer exchange, and the purification of monomeric r-oGH from aggregated complex was achieved in a single step using gel filtration chromatography. More than 60% of the initial inclusion body protein was refolded into a native-like conformation by the use of this procedure. The refolded protein was characterized by circular dichroism, fluorescence, SDS-PAGE, Western blotting, and radio receptor binding assay and found to be similar to native, pituitaryderived, ovine growth hormone.
Purification of large quantities of biologically active recombinant human growth hormone
Production and purification of human growth hormone using a simple method was studied in two recombinant Escherichia coli, D7-5 and C27-2 strains. The r-hGH was expressed in the form of inclusion body in a batch fermentation process and purified to 99% purity using a procedure based on acid precipitation of the host derived proteins and other impurities. The effect of the pH and host strain on purification of the r-hGH and efficiency of the procedure were evaluated. It was found that the optimum pH for precipitation of the host derived proteins was 4.9. The procedure was suitable for r-hGH purification from D7-5 stain but not from the other strain C27-2. The purity of > 99% and recovery of about 40% were obtained as shown by SDS-PAGE and Western blot analysis. The purified r-hGH was biologically active as judged by receptor assay with very low endotoxin content which could be suitable for therapeutic applications. This simple and cost effective production process could be useful for large scale production of recombinant hGH from specific strains.
High throughput purification of recombinant human growth hormone using radial flow chromatography
Protein Expression and Purification, 2009
Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. Using fed-batch fermentation process, around 670 mg/L of r-hGH was produced at a cell OD600 of 35. Cell lysis followed by detergent washing resulted in semi-purified inclusion bodies with more than 80% purity. Purified inclusion bodies were homogenous in preparation having an average size of 0.6 lm. Inclusion bodies were solubilized at pH 12 in presence of 2 M urea and refolded by pulsatile dilution. Refolded protein was purified with DEAE-anion exchange chromatography using both radial and axial flow column (50 ml bed volume each). Higher buffer flow rate (30 ml/min) in radial flow column helped in reducing the batch processing time for purification of refolded r-hGH. Radial column based purification resulted in high throughput recovery of diluted refolded r-hGH in comparison to axial column. More than 40% of inclusion body protein could be refolded into bioactive form using the above method in a single batch. Purified r-hGH was analyzed by mass spectroscopy and found to be bioactive by Nb2 cell line proliferation assay. Inclusion body enrichment, mild solubilization, pulsatile refolding and radial flow chromatography worked cooperatively to improve the overall recovery of bioactive protein from inclusion bodies.
International Journal of Peptide Research and Therapeutics, 2019
The 22 kDa of human growth hormone (hGH) is naturally produced and secreted by somatotrophic cells in the anterior part of the pituitary gland. The aim of this study was to clone, express, purify of hGH as fusion to the pelB leader in pET22b (+) plasmid and evaluate it's biological properties. The hGH polypeptide codon was optimized and subcloned. The recombinant hGH protein was purified by affinity chromatographic system against His-tag and the presence and accuracy of the purified products were evaluated by protein electrophoresis and Western blot. The biological activity of recombinant hGH protein was measured using ELISA assay. The results of sequencing of hGH gene confirmed the presence and proper placement of hGH gene and it's subcloning in the plasmid pET22b (+). The results of the protein electrophoresis and Western blot assays demonstrated that the expression accuracy of 22 kDa recombinant hGH. The results of Bradford spectroscopy assay showed that the recombinant hGH protein concentration was 1 g/l. The results of classical sandwich ELISA assay, in contrast to the specific antibodies, confirmed the bio-activity of the recombinant hGH protein in its targeting. Consequently, the results of this study showed that pelB leader has the ability to more accurately direct hGH to periplasmic space in Escherichia coli, and the conditions of oxidizing periplasmic space give rise to the correct folding of the protein in this space. Furthermore, the results of current study proved that using bioinformatics tools and combining them with laboratory data, could improve the recombinant hGH expression in E. coli, in addition to preserving bio-activity.
African Journal of Biotechnology, 2011
In this report, we describe the cloning, over-expression, efficient solubilization, purification and evaluation of bioactivity of camel growth hormone (cGH). The total cellular RNA was extracted from pituitary glands of freshly slaughtered animals and cDNA of cGH was synthesized by a pair of sequence specific primers with a product of 576 base pairs (bps). Amplicons was cloned into T/A cloning vector and positive clones were subjected to sequencing. After sequencing, cDNA was cloned in the prokaryotic expression vector system pET23b + . Conditions for cGH expression were optimized by varying the concentration of isopropyl-L-thio-β D-galactopyronoside (IPTG) and induction time. It was observed that 100 µM concentration of IPTG and 3 h post-induction produced the highest amount of cGH. Expressed GH was sequestered as inclusion bodies (IBs), and was therefore, solubilized using denaturant (urea) and detergents (SDS, CTAB, Tritin X-100, Tween-20). The best solubilization was obtained with 8.5 mM SDS in 100 mM Tris buffer at pH 8.5. The solubilized cGH was purified by gel filtration chromatography using Sephadex G-50 column. The purified protein was refolded by dialysis, analyzed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blot. Further biological activity of purified product was confirmed by efficient growth of rat Nb2 lymphoma cells. This study provided the method for the efficient solubilization of cGH (r-cGH) with comparable bioactivity with commercially available bovine growth hormone (bGH) and could be further used for solubilization of other proteins expressed in prokaryotic system.
PloS one, 2014
Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltosebinding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b9a9 domain of PDI (PDIb9a9), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18uC also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb9a9-, or Trx-tagged hGH expressed at 18uC in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, ,37 mg, ,12 mg, and ,7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb9a9-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.
The Biochemical journal, 1992
Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that th...
CHEMICAL & PHARMACEUTICAL BULLETIN, 1987
The efficient purification and characterization of the product of chemically synthesized human growth hormone (hGH) gene expressed in Escherichia coli are described. The product was purified from the cell lysates of the E. coli by means of ammonium sulfate precipitation, DE-52 chromatography, chromatofocusing chromatography and Ultrogel AcA 54 chromatography. The purified hGH gene product was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, non-denaturing polyacrylamide gel electrophoresis, gel electrofocusing and highperformance liquid chromatography (HPLC). The purified product and an authentic methionyl hGH (m-hGH) showed identical behavior in these systems. The structural features of the purified product were examined by means of amino acid composition analysis, NH2-terminal sequence analysis and tryptic peptide mapping. The experimental values of the amino acid composition of the purified product were in agreement with the theoretical values for m-hGH. Its NH2-terminal sequence (39 amino acid residues) was identical with that of the published sequence of hGH, except for an additional amino-terminal methionine residue immediately preceding phenylalanine at residue 1. The elution profile of the tryptic peptides of the purified product on HPLC was identical with that of authentic m-hGH. These elution profiles were nearly identical with that of a pituitaryderived hGH, with the exception of one peak due to NH2-terminal peptide. On the basis of these results, the purified product was identified as m-hGH. Keywords chemically synthesized gene; human growth hormone gene; human growth hormone; purified human growth hormone gene product
Microbial Cell Factories
Background Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. Results In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like...