• International Journal of Oral Biology
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• v.42 no.2
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• pp.71-78
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• 2017

BMP-2 is a well-known TGF-beta related growth factor, having a significant role in bone and cartilage formation. It has been employed to promote bone formation in some clinical trials, and to differentiate mesenchymal stem cells into osteoblasts. However, it is difficult to obtain this protein in its soluble and active form. hBMP-2 is expressed as an inclusion body in the bacterial system. To continuously supply hBMP-2 for research, we optimized the refolding of recombinant hBMP-2 expressed in E. coli, and established an efficient method by using detergent and alkali. Using a heparin column, the recombinant hBMP-2 was purified with the correct refolding. Although combinatorial refolding remarkably enhanced the solubility of the inclusion body, a higher yield of active dimer form of hBMP-2 was obtained from one-step refolding with detergent. The refolded recombinant hBMP-2 induced alkaline phosphatase activity in mouse myoblasts, at $ED_{50}$ of 300-480ng/ml. Furthermore, the expressions of osteogenic markers were upregulated in hPDLSCs and hDPSCs. Therefore, using the process described in this study, the refolded hBMP-2 might be cost-effectively useful for various differentiation experiments in a laboratory.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.2
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• pp.122-127
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• 2005

A size exclusion chromatography (SEC) process, in the presence of denaturant in the refolding buffer was developed to refold recombinant human $interferon-\gamma$ ($rhIFN-\gamma$) at a high concentration. The $rhlFN-\gamma$ was overexpressed in E. coli resulting in the formation of inactive inclusion bodies (IBs). The IBs were first solubilized in 8 M urea as the denaturant, and then the refolding process performed by decreasing the urea concentration on the SEC column to suppress protein aggregation. The effects of the urea concentration, protein loading mode and column height during the refolding step were investigated. The combination of the buffer-exchange effect of SEC and a moderate urea concentration in the refolding buffer resulted in an efficient route for producing correctly folded $rhIFN-\gamma$, with protein recovery of $67.1\%$ and specific activity up to $1.2\times10^7\;IU/mg$.

• Journal of the Korean Magnetic Resonance Society
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• v.23 no.4
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• pp.104-107
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• 2019

Many proteins are expressed as an insoluble form during the production using Escherichia coli (E. coli) system. Although various methods are applied to increase their amounts of soluble expression, refolding is the only feasible way to obtain a target protein in some cases. Moreover, protein NMR experiments require ¹³C/¹⁵N-labeled proteins that can only be obtained from E. coli systems in terms of cost and technical difficulty. The finding of appropriate refolding conditions for a target protein is a time-consuming process. In particular, it is very difficult to determine whether the refolded protein has a native structure, when a target protein has no enzymatic activity and its refolding yield is very low. Here, we showed that 1-dimensional ¹H-¹⁵N heteronuclear single quantum correlation (1D ¹H-¹⁵N HSQC) experiment can be efficiently used to screen an optimal condition for the refolding of a target protein by monitoring both the structure and concentration of the refolded protein.

• Journal of Life Science
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• v.17 no.2 s.82
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• pp.211-217
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• 2007

The goal of this study is to investigate effects of temperature and co-chaperonin requirement for in vitro protein refolding assisted by E. coli chaperone GroEL under permissive and nonpermissive temperature conditions. In vitro protein refolding of two denatured proteins was kinetically investigated under several conditions in the presence of GroEL. Effects of temperature and GroES-requirement on the process of prevention of protein aggregation and refolding of denatured protein were extensively monitored. We have found that E. coli GroEL chaperone system along with ATP is required for invitro refolding of unfolded polypeptide under nonpermissive temperature of $37^{\circ}C$. However, under permissive condition spontaneous refolding can occur due to lower temperature, which can competes with chaperone-mediated protein refolding via GroEL chaperone system. Thus, GroEL seemed to divert spontaneous refolding pathway of unfolded polypeptide toward chaperone-assisted refolding pathway, which is more efficient protein refolding pathway.

• KSBB Journal
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• v.16 no.5
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• pp.500-504
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• 2001

We scaled up a covalent immobilization system of urokinase to the activated Sepharose and used it repeatedly to cleava 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 6 M guanidine HCI after the cleavage reaction, the immobilized urokinase showed no activity probably becasue it was fully unfoled. However, as the denaturant was gradually removed from the column the immobilized urokinase fully regained its bioactivity, which indicated it was properly refolded into is natie 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 and 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.10 no.1
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• pp.75-80
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• 2000

An improved method of refolding recombinant human proinsulin from E. coli was presented. It was based on a two-stage stirred tank reactor in which denatured proinsulin-s-sulfonate was mixed instantaneously with a reaction buffer in the first stage reactor, and then fed to the second stage reactor. The mixture was stirred further for a total of 30h in the second stage reactor. In this system, unfavorable effects present due to the increase in reaction volume and protein concentration for protein refolding, which becomes significant in a large-scale operation, were avoided. Refolding yields of over 80% was obtained for achieving reaction volume of upto 50 l at protein concentration of 1 mg/ml. The optimum urea concentration was 1M. Refolding yield at the 1-1 reaction volume and protein concentration of 0.5mg/ml was increased about 2.5-fold, compared to that in a batch reactor. By increasing protein concentration in a two-stage refolding reaction, the cost for insulin production could be reduced, therefore, making this process economical.

• Korean Chemical Engineering Research
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• v.43 no.2
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• pp.187-201
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• 2005

Bioprocessing technologies utilizing 'biorecognition' between a solid matrix and a protein is being widely experimented as a means to replacing the conventional, solution-based technology. Frequently the matrices are chromatographic resins with specific functional groups exposed outside. Since the reactions of and interactions with the proteins occur as they are attached to the solid matrix, this 'solid-phase' processing has distinct advantages over the solution-phase technology. Solid-phase refolding of inclusion body proteins uses ion exchange resins to adsorb denaturant-dissolved inclusion body. As the denaturant is slowly removed from the micromoiety around the protein, it is refolded into a native, three-dimensional structure. Once the refolding is complete, the folded protein can be eluted by a conventional elution technique such as the salt-gradient. This concept was successfully extended to 'EBA (expanded bed adsorption)-mediated refolding,' in which the denaturant-dissolved inclusion body in whole cell homogenate is adsorbed to a Streamline resin while cell debris and other impurity proteins are removed by the EBA action. The adsorbed protein follows the same refolding steps. This solid-phase refolding process shows the potential to improve the refolding yield, reduce the number of processing steps and the processing volume and time, and thus improve the overall process economics significantly. In this paper, the experimental results of the solid-phase refolding technology applied to several biopharmaceutical proteins of various types are presented.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.197-203
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• 2001

A fusion protein, consisting of human epidermal growth factor as a recognition domain and human angiogenin as a toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation, probably due to the opposite surface charge due to vastly different pI values of each domain. Solid-phase refolding process exploiting ionic interactions between the solid matrix and the protein was tried, but the ionic binding yield was very low regardless of the resins and pH conditions used. To provide higher affinity toward the solid matrix, six lysine residues were tagged to the N -terminus of the hEGF domain When the cation exchange resins such as heparin- or CM-Sepharose were used as the matrix, the adsorption capacity increased 2.5-3 times and the subsequent refolding yield increased nearly IS times compared to the conventional process.

• BMB Reports
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• v.34 no.2
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• pp.102-108
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• 2001

A time-dependent folding process was used to determine whether or not protein disulfide isomerase (PDI) plays an important role in the maturation of nascent lactoferrin polypeptides. Interaction between lactoferrin and PDI was analyzed according to the co-immunoprecipitation of the two proteins. The results indicate that lactoferrin folding requires a significant interaction with PDI and its binding is relatively brief compared to other nascent polypeptides. The amount of lactoferrin interacting with PDI increases up to half a minute and sharply decreases beyond this time point. During the refolding process that follows reduction by DTT, lactoferrin polypeptides heavily interact with PDI and the interaction period was extended compared to the normal folding process. In terms of the temperature effect on PDI-lactoferrin interaction, PDI binds to lactoferrin polypeptides longer at a lower temperature (here, $25^{\circ}C$) than $37^{\circ}C$. The lactoferrin-PDI interaction was also studied in vitro. According to the in vitro experiment data, PDI was still functional in cell lysates assisting lactoferrin folding into the mature form. PDI interacts with lactoferrin polypeptides for an extended period during the folding in vitro. During the refolding process in vitro, intermolecular aggregates and refolding oligomers matured into a functional form after PDI binds to the lactoferrin. These results suggest that PDI provides a prolonged chaperoning activity in the refolding processes and that there appears to be a greater requirement for PDI chaperone activity in the refolding of lactoferrin polypeptides.

• BMB Reports
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• v.32 no.5
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• pp.486-491
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• 1999

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and propeptide-sequence regions of lignin peroxidase H2 (Lip H2) was overexpressed in Escherichia coli BL21 (DE3) to evaluate its catalytic characteristics and potential application as a pollution scavenger. All expressed proteins were aggregated in an inactive inclusion body, which might be due to inherent disulfide bonds. Active enzyme was obtained by refolding with glutathione-mediated oxidation in refolding solution containing $Ca^{2+}$, heme, and urea. Propeptide-sequence region was not processed as evidenced by N-terminal sequence analysis. Recombinant Lip H2 (rLip H2) had the same physical properties of the native protein but differed in the $K_{cat}$. Catalytic efficiency ($k_{cat}/K_m$) of rLip H2 was slightly higher than that of the native enzyme. In order to express an active protein, fusion systems with thioredoxin or Dsb A, which have disulfide isomerase activity, were used. The fused proteins expressed by the Dsb A fusion vector were aggregated, whereas half of the thioredoxin fusion proteins were recovered as a soluble form but still catalytically inactive. These results suggest that Lip H2 may not be expressed as an active enzyme in Escherichia coli although the activity can be recovered by in vitro refolding.