• Journal of the Korea Academia-Industrial cooperation Society
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• v.22 no.6
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• pp.542-549
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• 2021

The purpose of this study was to provide an economical and easy preparation method for recombinant human epidermal growth factor (rhEGF) without the need for an expensive enzyme to cleave the fusion part. However, the N-terminal fusion part is still useful for affinity chromatography. The hEGF is an important hormone in cell growth and proliferation in humans, and many studies on the expression and purification of this protein have been reported. In the present study, the hEGF gene was designed to be optimized with the E. coli codon usage preference and to contain Asn-Gly at the N-terminus of the protein. The gene was inserted into pRSET_A, an E. coli expression vector, and transformed into E. coli BL21 (DE3). The recombinant fusion protein was successfully co-expressed with pG-Tf2, a chaperone vector, in E. coli and purified by Ni-NTA column chromatography. The rhEGF was then released by hydroxylamine treatment and confirmed by SDS-PAGE. ELISA analysis showed that the activity of the free rhEGF was more than 92% similar to that of commercial EGF. The biological activity of the rhEGF was confirmed by a cell proliferation test with human skin fibroblasts.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.599-602
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• 2005

Truncated form of UBP1, an ubiquitin-specific protease of Saccharomyces cerevisiae, was overexpressed in Escherichia coli. The hexahistidine residue $(His_6)$ was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were $40^{\circ}C$ and pH 8.0, respectively.

• Proceedings of the PSK Conference
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• 2002.10a
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• pp.424.2-424.2
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• 2002

The in vivo release characteristics of rhGH-loaded PLGA microsphere prepared using a double emulsion process from hydrophilic 50:50 poly(D.L-lactide-co-glycolide) (PLGA) polymers were analyzed. This formulation showed particle size of ca 53.1$\mu\textrm{m}$ with high drug incorporation efficiency. To investigate in vivo release kinetics without the interference of formation of antibodies to rhGH in the experimental animals, the animals were immunosuppressed by treatment with Cyclosporin. (omitted)

• Archives of Pharmacal Research
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• v.25 no.5
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• pp.572-584
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• 2002

Rapid development in molecular biology and recent advancement in recombinant technology increase identification and commercialization of potential protein drugs. Traditional forms of administrations for the peptide and protein drugs often rely on their parenteral injection, since the bioavailability of these therapeutic agents is poor when administered nonparenterally. Tremendous efforts by numerous investigators in the world have been put to improve protein formulations and as a result, a few successful formulations have been developed including sustained-release human growth hormone. For a promising protein delivery technology, efficacy and safety are the first requirement to meet. However, these systems still require periodic injection and increase the incidence of patient compliance. The development of an oral dosage form that improves the absorption of peptide and especially protein drugs is the most desirable formulation but one of the greatest challenges in the pharmaceutical field. The major barriers to developing oral formulations for peptides and proteins are metabolic enzymes and impermeable mucosal tissues in the intestine. Furthermore, chemical and conformational instability of protein drugs is not a small issue in protein pharmaceuticals. Conventional pharmaceutical approaches to address these barriers, which have been successful with traditional organic drug molecules, have not been effective for peptide and protein formulations. It is likely that effective oral formulations for peptides and proteins will remain highly compound specific. A number of innovative oral drug delivery approaches have been recently developed, including the drug entrapment within small vesicles or their passage through the intestinal paracellular pathway. This review provides a summary of the novel approaches currently in progress in the protein oral delivery followed by factors affecting protein oral absorption.

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

• KSBB Journal
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• v.14 no.5
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• pp.593-599
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• 1999

In this study, optimal conditions to infect CD34 positive cells containing hematopoietic stem cells obtained from cord blood and bone marrow were found using two different retroviral vectors expressing human growth hormone (hGH) and $\beta$-galactosidase. CD34 positive cells were successfully infected with recombinant retroviruses only when the CD34 positive cells were co-cultured with packaging cells secreting recombinant retroviruses. To find the highest infection efficiency for the gene transfer, CD34 positive cells from cord blood were co-cultured with packaging cells secreting recombinant retroviruses encoding E. coli lacZ gene. The highest infection efficiency was obtained when CD34 positive cells were cultured for 3 days, and then co-culturing was done for another 2 days. When CD34 positive cells from bone marrow were co-cultured with packaging cells secreting recombinant retroviruses encoding hGH gene, the maximum amount of hGH was also secreted at the same conditions found above, i.e. 3 days of culture and 2 days of co-culture. These results show that there are optimal conditions for the gene transfer into hematopoietic stem cells regardless of sources of target cells or retroviral vectors used to infect.

• Reproductive and Developmental Biology
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• v.34 no.4
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• pp.289-297
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• 2010

Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the $105^{th}$ amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MIT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.

• KSBB Journal
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• v.16 no.2
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• pp.146-152
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• 2001

To avoid the intrinsic problem of aggregation associated with the traditional solution-phase refolding process, we propose a solid-phase refolding method integrated with expanded bed adsorption chromatography. The model protein used was a fusion protein of recombinant human growth hormone and a glutathione S transferase fragment. It was demonstrated that the EBA-mediated refolding technique could simultaneously remove cellular debris and directly renature the fusion protein inclusion bodies in the cell homogenate with much higher yields and less agregation. To demonstrate the applicability of the method, we successfully tested the three representative types of starting materials, i. e., rhGH monomer, washed inclusion bodies, and the E. coli homogenate. This direct and simplified refolding process could also reduce the number of renaturation steps required and allow refolding at a higher concentration, at approximately 2 mg fusion protein per ml of resin. To the best of our knowledge, it is the first approach that has combined the solid-phase refolding method with expanded bed chromatography.