• Korean Journal of Veterinary Research
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• v.49 no.1
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• pp.39-44
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• 2009

Transmission of avian viruses both bird-to-bird and from birds to non-avian species is a major health concern. Newcastle disease virus (NDV) is an economically important avian virus that poses substantial risks to the poultry industry. Rapid and sensitive diagnostic methods, such as the enzymelinked immunosorbent assay (ELISA), are required to track such infections. To develop an ELISA for detecting anti-NDV antibody in avian sera, the nucleocapsid protein (NCP) gene of the NDV La Sota strain was cloned and expressed in Escherichia coli and the 513-amino acid recombinant NCP was purified by Ni-NTA affinity chromatography. To evaluate its ability to replace NDV whole virus antigen as a coating antigen, NCP-coated and whole NDV-coated ELISAs were tested and compared using a panel of NDV positive antisera from chickens. Results using purified NCP were highly correlated with those obtained using whole NDV (r= 0.927), demonstrating that recombinant NCP expressed in Escherichia coli is a suitable substitute antigen for whole NDV in a diagnostic ELISA.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1126-1134
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• 2024

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.210-213
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• 2005

The suppression of apoptosis during the cell culture might increase recombinant protein production. In the present study, the effects of anti-apoptotic agents on the apoptosis of recombinant Chinese Hamster Ovary cells and the production of Iduronate 2-sulphatase(IDS) were investigated Cell density slightly increased when $2{\mu}M$ of EGCG and $10{\mu}g/mL$ of STR-G were added to culture medium after two days. It was observed that the percentage of apoptotic cells was decreased in the culture with STR-G, and Bcl-2 expression level was enhanced in both culture with STR-G and EGCG. These results suggest that G418 and EGCG are effective anti-apoptotic agents for increasing the productivity of IDS with recombinant CHO-DG44.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.13-16
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• 2000

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed in Escberichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced in E. coli cytoplasm were easily dissolved by simple alkaline pH shift $(8\rightarrow12\rightarrow8)$. Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.

• Journal of Periodontal and Implant Science
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• v.37 no.1
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• pp.35-44
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• 2007

Periodontal ligament (PDL) is the connective tissue located between the tooth root and alveolar bone. In a previous study, PDLs22 was isolated as a PDL-specific gene by using subtractive hybrid-ization between cultured PDL fibroblasts and gingival fibroblasts. It was also suggested that PDLs22 plays important roles in the development, differentiation and maintenance of periodontal tissues. However, little is known about functional study of PDLs22 using recombinant protein in PDL fibroblast differentiation and periodontium formation. In this study, in order to produce the PDLs22 recombinat protein, PDLs22 expression vector were constructed and expressed its protein in various host cell and temperature conditions. The results were as follows: 1. PDLs22 protein was not strongly expressed In the induction system using pRSET-PDLs22 construct. 2. When the BL21(DE3) pLysS was used as a expression host, PDLS22 protein was strongly ex-pressed in the induction system using pHCEIIBNd-PDLs22 construct. 3. The PDLs22 protein was recognized at a molecular weight of 28 kDa in western blots. 4. Almost of the expressed PDLs22 protein was not soluble and observed like as inclusion body. 5. The protein solubility was not improved after modification of induction time and temperature during PDLs22 protein production. In this study, the system for the PDLs22 protein production was connstructed. However, the re-results suggest that further studies will be needed to produce the considerable amount of PDLs22 re-combinat protein, which can use for the periodontal regeneration.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.153-159
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• 2008

The limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.

• Journal of Periodontal and Implant Science
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• v.38 no.1
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• pp.41-50
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• 2008

Purpose: Bone morphogenetic protein-2(BMP-2) has been shown to possess significant osteoinducitve potential. There have been attempts to overcome a limitation of mass production, and economical efficiency of BMP. The aim of this study was to produce recombinant human BMP-2(rhBMP-2) from E. coli in a large scale and evaluate its biological activity. Materials and Methods: The E.coli strain BL21(DE3) was used as a host for rhBMP-2 production. Dimerized rhBMP-2 was purified by affinity chromatography using Heparin column. To determine the physicochemical properties of the rhBMP-2 expressed in E. coli, we examined the HPLC profile and performed Western blot analysis. The effect of the purified rhBMP-2 dimer on osteoblast differentiation was examined by alkaline phosphatase (ALP) activity and representing morphological change using C2C12 cell. Results: E. coli was genetically engineered to produce rhBMP-2 in a non-active aggregated form. We have established a method which involves refolding and purifying a folded rhBMP-2 dimer from non-active aggregates. The purified rhBMP-2 homodimer was characterized by SDS-PAGE as molecular weight of about 28kDa and eluted at 34% acetonitrile, 13.27 min(retention time) in the HPLC profile and detected at Western blot. The purified rhBMP-2 dimer stimulated ALP activity and induced the transformation from myogenic differentiation to osteogenic differentiation. Conclusion: rhBMP-2 was produced in E. coli using genetic engineering. The purified rhBMP-2 dimer stimulated ALP activity and induced the osteogenic differentiation of C2C12 cells.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.313-316
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• 2000

When 3 recombinant Chinese hamster ovary (rCHO) cell lines, CHO/dhfr-B-22-4, $CS13-1.00^{\ast}$ and $CSl3-0.02^{\ast}$, were cultivated in hyperosmolar media resulting from NaCl addition, their specific foreign protein productivity increased with medium osmolality. Glycine betaine was found to have a strong osmoprotective effect on all 3 rCHO cell lines. Inclusion of 15 mM glycine betaine in hyperosmolar medim enabled rCHO cell lines to grow at 557-573 mOsm/kg where they could not grow in the absence of glycine betaine. However, effect of glycine betaine inclusion in hyperomolar medium on foreign protein production differed among rCHO cell lines. CHO/dhfr-B22-4 cells retained enhanced specific human thrombopoietin (hTPO) productivity in the presence of glycine betaine, and thereby, the maximum hTPO titer obtained at 573 mOsm/kg was increased by 72% over that obtained in the control culture with physiological osmolality (292 mOsm/kg). On the other hand, enhanced specific antibody productivity of $CSl3-1.00^{\ast}$ and $CSl3-0.02^{\ast}$ at elevated osmolality decreased significantly in the presence of glycine betaine at a cost of the recovery of cell growth. As a result, the maximum antibody titer at 557 mOsm/kg was similar to that obtained in the control culture with physiological osmolality. Taken together, efficacy of the simultanous use of hyperosmotic pressure and glycine betaine as a means to improve foreign protein production was variable among different rCHO cell lines.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1742-1750
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• 2015

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at $15^{\circ}C$, whereas the reaction at $25^{\circ}C$ was faster than that at $15^{\circ}C$. The refolding yield at $15^{\circ}C$ was 17% higher than that at $25^{\circ}C$. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at $15^{\circ}C$ for 16 h. The maximum refolding yield was 74.8% at $15^{\circ}C$ with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.