title: On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA.
abstract: DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.
authors.affiliation: Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, PR China.
authors.forename: Man
authors.initials: M
authors.lastname: Luo
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Guan
authors.forename: Shan-Jing
authors.initials: SJ
authors.lastname: Yao
mesh_terms: D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding
chemical_list: D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D009113:Muramidase; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli