Urea-Gradient Protein Refolding in Size Exclusion Chromatography

Wang, Chaozhan; Chen, Yan

doi:10.2174/138920110791112004

Cited by 4 publications

(3 citation statements)

References 11 publications

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“…Various strategies have been devised to overcome this drawback. Refolding of urea-denatured proteins has been carried out using size exclusion chromatography (24,36). Attempts have also been made to increase the fraction of soluble protein expressed in Escherichia coli by decreasing the growth temperature to between 28 and 30°C (32).…”

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Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Prasad

Khadatare

Roy

2011

Appl Environ Microbiol

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The recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies in Escherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.Overexpression of proteins in heterologous systems often results in the formation of inclusion bodies. These electronrefractile particles are formed because of the failure of prokaryotic folding machinery to correctly fold the nascent polypeptide chain, increasing its local concentration in the cytosol. The most common strategy to recover active proteins from these particles is by denaturing them in the presence of chaotropes and refolding them to the native conformation by the gradual removal of the denaturant. This process results in high loss in protein yield since the renaturation efficiency of most proteins is not very high.

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confidence: 99%

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Prasad

Khadatare

Roy

2011

Appl Environ Microbiol

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“…The results indicated that UGSEC yielded a higher activity recovery than the other two methods. 29 Additionally, the difference in activity recovery between gradient refolding and the other two methods was more significant for higher initial protein concentrations. 30,31 In this study, we used a size exclusion chromatography-based approach for in vitro refolding recombinant teriparatide and simulated the elution behavior inside the size exclusion chromatography column.…”

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confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mechanistic Modeling of Size Exclusion Chromatography-Assisted In Vitro Refolding of the Recombinant Biosimilar Teriparatide (PTH-34)

Ughade,

Rana,

Nadeem

et al. 2024

ACS Omega

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In vitro protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using Escherichia coli as host cells. This study is focused on designing size exclusion chromatography-assisted in vitro refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and in vitro refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on in vitro refolding of recombinant teriparatide was studied using the Box−Behnken design. Size exclusion chromatography (SEC)-assisted in vitro refolding was performed at 8 °C at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post in vitro refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.

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Production and Purification of Recombinant Proteins from Escherichia coli

Tripathi

2016

ChemBioEng Reviews

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Development of successful production strategies for recombinant proteins is a challenging task due to problems associated with the scale‐up of bioprocesses. Recent developments in fermentation technology have provided a suitable platform to produce bioproducts for the development of diagnostics, therapeutics and vaccines for bacterial, viral and other diseases. A bacterial system is the commonly used expression system for production of recombinant products such as proteins, enzymes, and antibodies. Escherichia coli is one of the most commonly used bacterial hosts for the production of these products. A variety of recombinant proteins has been produced and cultivation studies have been carried out at small scale. However, the majority of the recombinant proteins are not amenable directly to large scale production processes due to various factors. To meet the growing demand of potential bioproducts, there is a need to produce these products in large quantity employing fermentation processes. Development of a suitable purification strategy is yet another important step to recover biologically active products. The present paper reviews the current developments in production and purification of recombinant products from E. coli. The bioproducts obtained after fermentation and purification processes may fulfill the requirements for the development of various diagnostics, therapeutics, or prophylactic agents against different kinds of diseases.

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Urea-Gradient Protein Refolding in Size Exclusion Chromatography

Cited by 4 publications

References 11 publications

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

Mechanistic Modeling of Size Exclusion Chromatography-Assisted In Vitro Refolding of the Recombinant Biosimilar Teriparatide (PTH-34)

Production and Purification of Recombinant Proteins from Escherichia coli

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