Immobilization of Protein Involving Disulfide Bond for Quantitative Evaluation of Refolding Media

JOURNAL OF CHEMICAL ENGINEERING OF JAPAN

Mikio Kitora¹  Tomoya Sakai¹  Daisuke Nohara¹  Yoshiko Shoda¹ 

[1]Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City University

关键词: Protein Refolding;    Refolding Media Evaluation;    Disulfide Bond;    Immobilized Enzyme Preparation;   

DOI  :  10.1252/jcej.35.908

来源: Maruzen Company Ltd

【 摘 要 】

References(22)In order to select adequate solvents for protein refolding, immobilized preparation of a given protein that is covalently fixed on the surface of support materials, has been employed. This method is greatly useful for selection of solvents appropriate to refolding itself of the protein molecule, excluding unfavorable intermolecular interactions such as aggregation and autoproteolysis that emerge concurrently during the refolding. The purpose of this work is to obtain the preparation that warrants the constant recovered activity in the same denaturation/renaturation procedure. Such immobilized preparation is necessary for quantitative estimation of ability of refolding solvents. The globular proteins employed were subtilisin BPN’, ribonuclease A and Streptomyces griseus trypsin. In the case of such a protein as subtilisin possessing no disulfide bond in the molecule, agarose beads which have been widely used exhibited high retained activity and the constant recovered activity in the repeated denaturation/renaturation procedures, and revealed to be excellent as a support material. However, agarose gels were not suitable in the case of the other enzymes possessing disulfide bonds. This is probably attributable to involvement of reduction and oxidation reactions in the procedure applied to the latter case. Investigation of various support materials other than agarose beads revealed that the stable immobilized preparation of disulfide-involving protein could be established by use of glass beads that are resistant against oxidation and reduction with relatively high retained activity.

【 授权许可】

Unknown