Synthesis and sequence-specific proteolysis of a hybrid protein (colicin A :: growth hormone releasing factor) produced in Escherichia coli (original) (raw)
Related papers
European Journal of Biochemistry, 1987
A recombinant gene comprising phoS (the gene for the phosphate-binding protein PhoS) fused to a synthetic gene for a modified human growth-hormone-releasing factor (mhGRF) has been constructed. This gene was highly expressed in cells growing under conditions of phosphate starvation. Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS-mhGRF). Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre-PhoE and pre-OmpA. The pre-PhoS-mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation. This protein was purified. Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein. Thus cyanogen bromide treatment allowed the isolation of pure mhGRF. The yield obtained is about of 1 mg/l culture.
BMC Biotechnology, 2021
Background: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISAbased method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.
High-level production of human growth hormone in Escherichia coli by a simple recombinant process
Journal of Biotechnology, 1998
Procedures have been devised for producing in Escherichia coli high yields of purified recombinant human growth hormone (hGH), by utilizing N-terminal pentapeptide sequence of human tumor necrosis factor-alpha, histidine tag and enterokinase cleavage site as a fusion partner. The fusion protein was produced as a soluble protein at the beginning of gene expression, but progressively became insoluble in Escherichia coli cytoplasm. The insoluble protein was solubilized by simple alkaline pH shift and purified to near homogeneity by Ni 2 + -chelated affinity chromatography. Following specific enterokinase cleavage, the recombinant hGH was purified by one-step anion exchange chromatography. The ease and speed of this recombinant process, as well as the high productivity, makes it adaptable to the large-scale production of hGH. Moreover, the highly efficient fusion partner could be applied to the production of other therapeutically important proteins.
1998
Procedures have been devised for producing in Escherichia coli high yields of purified recombinant human growth hormone (hGH), by utilizing N-terminal pentapeptide sequence of human tumor necrosis factor-alpha, histidine tag and enterokinase cleavage site as a fusion partner. The fusion protein was produced as a soluble protein at the beginning of gene expression, but progressively became insoluble in Escherichia coli cytoplasm. The insoluble protein was solubilized by simple alkaline pH shift and purified to near homogeneity by Ni 2 +-chelated affinity chromatography. Following specific enterokinase cleavage, the recombinant hGH was purified by one-step anion exchange chromatography. The ease and speed of this recombinant process, as well as the high productivity, makes it adaptable to the large-scale production of hGH. Moreover, the highly efficient fusion partner could be applied to the production of other therapeutically important proteins.
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.
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
2005
A gene encoding the mature form of the human growth hormone (hGH) was fused to the secretion signal coding sequence, pelB, this hybrid gene was expressed in E. coli under the transcriptional control of bacteriophage T7\lac promote\operator system. Studying the periplasmic protein pattern of the recombinant bacteria, showed that the recombinant bacteria allowed a partially succeeded secretion of hGH into the periplasmic space. Remaining of unprocessed pelB::hGH fusion-protein was also observed in the E. coli cytoplasmic space. Comparing to IPTG induction, we have also used lactose as natural inducer for over-expression of rhGH in the present system. Our results demonstrated that lactose is as effective as IPTG for inducing expression of recombinant hGH. Introduction Variation of parameters involved in expression of recombinant protein is a phenomenon commonly observed in designing strategies for genetically engineering proteins. Different permutation and combinations of vectors, prom...
In order to study the periplasmic expression of human growth hormone (hGH) in Escherichia coli, the related cDNA was inserted in two expression plasmids carrying pelB signal peptide, one with lac bacterial promoter and the other with a bacteriophage T7-based promoter. The recombinant plasmids were moved to TG1 and BL21 strains of E. coli, respectively. To induce the expression systems, IPTG and its natural analog, lactose, were examined. Results show the over-expression of recombinant hGH (rhGH) in the T7-based system which is much higher than that of the lac-regulated system. In both systems, a fraction of the hGH, produced by recombinant bacteria, remains in the cytoplasm as pre-protein and the rest is transferred to the periplasmic space as mature protein. Any decline in the expression level did not lead to a complete processing and transport of mature hGH to the periplasmic space of the bacteria. It is suggested that, for an efficient expression of rhGH in the periplasmic space of E. coli, a combined approach including application of a suitable signal peptide, solubility of the over-expressed protein in cytoplasm in addition to the optimization of the bacterial growth and inducing conditions should be considered.