• BMB Reports
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• v.32 no.3
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• pp.306-310
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• 1999

The partial cDNA of the MT-c clone encoding snake venom metalloprotease was subcloned and expressed in E. coli. The expressed metalloprotease was purified by affinity chromatography in the presence of urea, and then successfully refolded into its functional form, retaining metalloprotease activity that hydrolyzes fibrinogen. The simple and convenient refolding strategy established in this work was highly efficient in recovering the recombinant enzyme activity. Experimental evidence suggests that the C-terminal amino acid stretch of 16 residues is a critical sequence for proper folding of the metalloprotease domain.

• KSBB Journal
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• v.18 no.6
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• pp.500-505
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• 2003

‘LK (lipoprotein kringle) 68’is a polypeptide of a modified ansiostatin consisting of three kringle structures that might be clinically useful as a potential cancer therapeutics. It can be produced by overexpressing it as inclusion body in recombinant E. coli. In this study, solid-phase refolding processes using packed bed adsorption (PBA) and expanded bed adsorption (EBA) column were carried out to compare their refolding yields with that of the conventional, solution-phase refolding process, For the solution-phase and the PBA-mediated processes employing Q-Sepharose, washed inclusion body was used as the starting material, whereas both washed inclusion body and E. coli homogenate were used for the EBA-mediated process employing streamline DEAE. On the final recovery LK68 per unit mass of wet cell basis, the EBA- and PBA-mediated processes showed about 2.7- and 1.5-fold higher yields, respectively, than the solution-phase refolding method. The solid-phase refolded LK68 demonstrated the same Iysine binding bioactivity and the retention time in the RP-and SEC-HPLC as those of the native protein.

• 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.17 no.2
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• pp.153-157
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• 2002

A fusion protein, consisting of a human epidermal growth factor as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as an inclusion body in recombinant E. coli, yet when the conventional solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably because of the opposite surface charge resulting from the vastly different pl values of each domain. Accordingly the solid-phase refolding process, which exploits the ionic interactions between a solid matrix and the protein, was tried, however the ionic binding yield was also very low regardless of the resins and pH conditions used. Therefore, to provide a higher affinity toward the solid matrix, six Iysine residues were tagged to the N-terminus of the hEGF domain. When cation exchange resins, such as heparin- or CM-Sepharose, were used as the matrix, the adsorption capacity increased 2.5~3-fold and the subsequent refolding yield increased nearly 15-fold compared to the conventional process. A similat result was also obtained when an Ni-NTA metal affinity resin was used.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.632-637
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• 2000

This research was undertaken to restore the biological activity of cyclodextrin glucanotransferase (CGTase) of Bacillus macerans origin expressed as inclusion bodies in recombinant Escherichia coli. The optimum concentration of urea used as a denaturant was 8 M. The supplementation of 0.5 M urea into a dialysis buffer increased the refolding efficiency by preventing any protein aggregation. The influence of the protein concentration, temperature, and pH were also investigated. The protein concentration was found to be the most important factor in the refolding efficiency. The optimum temperature was 15-$25^{\circ}C$ and the optimum pH was 6.0. The maximum specific activity of the CGTase refolded under the optimum conditions was 92.2 U/mg, corresponding to 72% of the native CGTase. A comparison of the secondary structure between the native and the refolded CGTase showed that the relative ratio of the $\alpha$-helix content in the native to the refolded CGTase was 1:0.82.

• KSBB Journal
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• v.20 no.5 s.94
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• pp.338-340
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• 2005

Covalent modification of a protein with polyethylene glycol (PEG) has become one of the most widely used and well established drug enhancement strategies in the biopharmaceutical industry. The general benefits enjoyed by PEGylation, such as prolonged serum half-lives or reduced immunogenicity in vivo, are well known. By now the PEGylation process has been performed with purified proteins, and it is required to recover the desired PEGylate by a multi-step purification process. The ultimate aim of our research is to develop an integrated process of PEGylation and in vitro refolding starting with inclusion body material. For this, we investigated the feasibility that a protein could be PEGylated under a denaturing condition and also the PEGylated proteins could be refolded correctly. Using lipase as a model protein, we found that it was PEGylated in the presence of 8M urea and that the PEG molecules covalently attached to lipase did not appear to hinder its refolding.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.85-88
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• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleave a fusion protein consisting of human growth hormone and GST fragment. After scale up from 6 ml to 250 ml, the column system still demonstrated basically the same performance in terms of urokinase immobilization and fusion protein cleavage. When the column was washed with 6M guanidine HCl after the cleavage reaction. the immobilized urokinase showed no activity probably because it was fully unfolded. However. as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity. which indicated it was properly refolded into its native conformation as covalently attached to the solid matrix. After 20 cycles of this 'solid-phase unfolding/refolding', the immobilized urokinase maintained approx. 80% of the initial bioactivity. This method provides an efficient protocol to apply the solid-phase refolding technique to improve the longevity of immobilized enzyme columns.

• Journal of Microbiology and Biotechnology
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• v.6 no.4
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• pp.225-231
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• 1996

The production of recombinant proteins in Escherichia coli often leads to the formation of an intracellular inclusion body. Key process steps that can determine the economics of large-scale protein production from inclusion bodies are fermentation, inclusion body recovery, and protein refolding. Compared with protein refolding and fermentation, inclusion body recovery has received scant research attention. Nevertheless, it can control the final product yield and hence process cost for some products. Optimal separation of inclusion bodies and cell debris can also aid subsequent operations by removing contaminant particulates that foul chromatographic resins and contain antigenic pyrogens. In this review, the properties of inclusion bodies and cellular debris are therefore examined. Attempts to optimise the centrifugal separation of inclusion bodies and debris are also discussed.

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.365-368
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• 1998

The aklavinone 11-hydroxylase which was overexpressed using T7 promoter in E. coli could be detected in SDS-PAGE only in insoluble precipitate without any detectable enzyme activity. The insoluble enzyme was solubilized in 6M guanidine$.$HCl solution and their refolding ability was tested under various conditions. When the enzymatic activity was checked by the bioconversion experiment, stepwise dialysis against 6M, 3M, 1M guanidine$.$HCl and finally 100 mM potassium phosphate buffer of the solubilized protein gave the best bioconversion efficiency. The aklavinone 11-hydroxylase showed its enzymatic activity in the reaction buffer containing NADPH with vigorous shaking. The enzymatic activity was lost during partial purification and regained by the addition of crude extract of S. lividans in the reaction mixture. This effect was confirmed to due to some low-molecular weight component(s) in the crude extract, because the addition of dialyzed crude extract could not recover the enzymatic activity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.237-243
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• 2001

Substantial progress has been made towards understanding the folding mechanisms of proteins in virto and in vivo even though the general rules governing such folding events remain unknown. This paper reviews current folding models along with experimental approaches used to elucidate the folding pathways. Protein misfolding is discussed in relation to disease states, such as amyloidosis, and the recent findings on the mechanism of converting normally soluble proteins into amyloid fibrils through the formation of intermediates provide an insight into understanding the pathogenesis of amyloid formation and possible cules for the development of therapeutic treatments. Finally, some commonly adopted refolding strategies developed over the part decade are summarized.