• Title, Summary, Keyword: Refolding

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Improved Large-Scale Refolding Techniques for Inclusion Body Proteins (내포체 단백질의 개선된 대규모 재접힘 기술)

  • 김인호;정봉현
    • KSBB Journal
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    • v.16 no.1
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    • pp.11-14
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    • 2001
  • Techniques for protein refolding from inclusion body are discussed in view of its engineering application to large scale protein purification. Among the techniques, dilution and dialysis are mainly utilized due to simple operation. Membrane reactor, gel filtration chromatography, and continuous tank operation are emerging tools for their process-scale possibility in refolding. Reaction engineering approaches could be used to analyze the kinetic behaviour in the process scale refolding reactor. The kinetic analysis is helpful in the optimization of refolding yield in the refolding reactor.

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Protein Aggregation and Adsorption upon In vitro Refolding of Recombinant Pseudomonas Lipase

  • Lee, Young-Phil;Rhee, Joon-Shick
    • Journal of Microbiology and Biotechnology
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    • v.6 no.6
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    • pp.456-460
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    • 1996
  • Recombinant Pseudomonas lipase was used to study protein aggregation and adsorption upon in vitro refolding. Protein adsorption as well as aggregation was responsible for major side reactions upon in vitro refolding as a function of protein concentration. The optimal range of protein concentration was determined by the relative contribution of protein aggregation and adsorption. Above the optimal range, the yield of active lipase inversely correlated with protein aggregation, showing a competition between folding and aggregation. However, adsorption of protein rather than protein aggregation is thought to contribute as a major side reaction of the refolding process at sub-optimal concentrations at which the formation of aggregates should be more reduced. Protein aggregation was influenced by the amount of guanidine hydrochloride in the refolding solvent. The refolding temperature was a critical factor determining the extent of protein aggregation. The refolding yield was also affected by the dilution fold and dilution mode, which suggests that the refolding process might kinetically compete with the rate of mixing.

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Large-Scale Refolding and Enzyme Reaction of Human Preproinsulin for Production of Human Insulin

  • Kim, Chang-Kyu;Lee, Seung-Bae;Son, Young-Jin
    • 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.

In Vitro Formation of Active Carboxypeptidase Y from Pro-Carboxypeptidase Y Inclusion Bodies by Fed-Batch Operation

  • Hahm, Moon-Sun;Chung, Bong-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.11 no.5
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    • pp.887-889
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    • 2001
  • The gene encoding yeast pro-carboxypeptidase Y (pro-CPY) has been cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was accumulated as cytoplasmic insoluble aggregates. In our previous study, active CPY was obtained by renaturation of entirely denatured pro-CPY followed by in vitro proteolytic processing with proteinase K along with the activation process. The same refolding process was performed to produce an active CPY from pro-CPY inclusion bodies with renaturation buffers containing proteinase K at different concentrations. The refolding efficiency decreased from $25\%\;to\;2\%$ in the renaturation buffers containing proteinase K at concentrations of $60{\mu}g/ml\;and\;0.6{\mu}g/mi$, respectively. In an attempt to increase the refolding efficiency with a lesser amount of proteinase K, a novel fed-batch refolding process was developed. In a fed-batch refolding, 99 ml of the renaturation buffer containing pro-CPY was gradually added into 1 ml of the renaturation buffer containing $60{\mu}g/ml$ of proteinase K to give a final proteinase K concentration of $0.6{\mu}g/ml$. The fed-batch refolding process resulted in a refolding efficiency of $18\%$, which corresponded to a 9-fold increase over that ($2\%$) in the batch process.

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Refolding of Fusion Ferritin by Gel Filtration Chromatography(GFC)

  • Kim, Hyung-Won;Kim, In-Ho
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.10 no.6
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    • pp.500-504
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    • 2005
  • Fusion ferritin (heavy chain ferritin, $F_H+$ light chain ferritin, $F_L$), an iron-binding protein, was primarily purified from recombinant Escherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, and L-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclusion bodies (IBs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.

In Vitro Refolding of Inclusion Body Proteins Directly from E. coli Cell Homogenate in Expanded Bed Adsorption Chromatography (Expanded Bed Adsorption 크로마토그래피를 사용하여 재조합 E. coli 세포 파쇄액으로부터 내포체 단백질을 직접 재접힘하는 공정)

  • 조태훈;서창우;이은규
    • 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.

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Improved Refolding of Recombinant Human Proinsulin from Escherichia coli in a Two-stage Reactor System

  • Phue, Je-Nie;Oh, Sung-Jin;Son, Young-Jin;Kim, Yong-In;Kim, Kyung-Hwan;Kim, Jung-Woo;Hong, Chung-Il;Chung, In-Sik;Hahn, Tae-Ryong
    • 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.

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Comparative Analysis of Dissolution and Refolding Processes for Inclusion Body Protein Renaturation (내포체 단백질 재생을 위한 용해 및 재접힘공정의 비교분석)

  • 김창성;김윤하;이은규
    • KSBB Journal
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    • v.13 no.2
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    • pp.133-140
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    • 1998
  • Using rlFN-$\alpha$ and rhGH as the model proteins, the refolding performances of the published processes were evaluated and compared. Key engineering parameters such as the type of denaturant and this concentration, protein concentration in the refolding buffer, and pH and ionic strength of the buffer were experimentally investigated. Furthermore, the role of a co-solvent of surfactant type in aggregation reduction was also studied. Of the denaturants tested (8M urea, 6M guanidine HCI, 0.5% SDS), SDS at alkaline pH (9.5) and ambient temperature gave the highest recovery yield. The SDS process was effective in the refolding of observed where dissolution proceeded better under lower strength (10 mM) but aggregation was suppressed under higher strength (>50 mM.) When PEG-4000 and/or Tween were added as co-solvent or refolding-enhancing additive, 1.6-2 times higher yield was realized. The‘masking’of the hyrophobic patches located on the surface of the protein with the surfactant molecules was believed to be responsible for the considerable reduction in aggregation during refolding.

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Solid-phase Refolding of Poly-lysine Tagged Fusion Protein of hEGF and Angiogenin

  • Park Sang Joong;Ryu Kang;Suh Chang Woo;Chai Young Gyu;Kwon Oh Byung;Park Seung Kook;Lee Eun Kyu
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.7 no.1
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    • pp.1-5
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    • 2002
  • A fusion protein, consisting of a human epidermal growth factor (hEGF) 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 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. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

Refolding of Proteins at High Concentration by Size Exclusion Chromatography

  • Guan, Yixin;Gao, Yonggui;Yao, Shanjing;Cho, Man-Gi
    • Proceedings of the Korean Society of Life Science Conference
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    • pp.9-17
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    • 2002
  • Renaturation of Lysozyme by size exclusion chromatography(SEC) to improve yield as well as the initial and final protein concentration has been studied in detail, Although urea decreases the rate of proteins refolding, it can suppress protein aggregation to sustain pathway of correct refolding at high protein concentration, and there existed an optimum urea concentration in renaturation buffer. Lysozyme was successfully refolded from initial protein concentration of up to 100mg/m1 by SEC, the yield was more than 40%. And the refolding of Interferon-${\gamma}$ was further investigated.

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