Inhibition of aggregation by media selection, sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B Zhenyu Gu a , Xiaonan Zhu b , Shaowei Ni a , Haimeng Zhou a , Zhiguo Su b, * a Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China b State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, PR China Abstract The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding. D 2003 Published by Elsevier Science B.V. Keywords: Aggregation; Refolding; Size exclusion chromatography; Resolution; Bovine carbonic anhydrase B 1. Introduction Ever since Anfinson [1] reported the significant discovery of the spontaneous refolding of ribonuclease, the protein refolding has been a subject of intensive investigation in the field of biotechnology. With the advent of genetic engineering, many valuable proteins were expressed in Escherichia coli in order to be mass- 0165-022X/03/$ - see front matter D 2003 Published by Elsevier Science B.V. doi:10.1016/S0165-022X(03)00057-5 * Corresponding author. Tel.: +86-10-82627095; fax: +86-10-62561822. E-mail address: zgsu@home.ipe.ac.cn (Z. Su). www.elsevier.com/locate/jbbm J. Biochem. Biophys. Methods 56 (2003) 165 – 175