Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Abstract

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.

The Ribosome Can Prevent Aggregation of Partially Folded Protein Intermediates: Studies Using the Escherichia coli Ribosome

“…The purpose of the present study was to investigate the ability of E.coli ribosome and the domain V of its 23S rRNA to interact with a partially folded intermediate state of proteins and influence their aggregation and reactivation under refolding conditions. Our studies were performed on the proteins a) bovine carbonic anhydrase II (BCAII) which under mild denaturing conditions assumes an equilibrium molten globule state [4] and b) chicken egg white lysozyme that forms a hydrophobically collapsed state at the onset of its folding process that have properties characteristic of equilibrium molten globule [5]–[9].…”

Extracellular production of functional single-chain variable fragment against aflatoxin B1usingEscherichia coli

“…; Wang et al . ). The addition of 0·25% Triton X‐100 to the culture medium upon IPTG induction resulted in a slight decrease (7%) of colony forming units, indicating that cell lysis by Triton X‐100 was not significant at this concentration (Fig.…”

Refolding of biotech therapeutic proteins expressed in bacteria: review

“…Wang et al . used the combination of chaperone and IEC (denoted as AMC‐IEC) to refold urea‐denatured/dithiothreitol‐reduced lysozyme . Factors influencing the refolding of lysozyme included urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent were optimized.…”