title: Optimization of dilution refolding conditions for a camelid single domain antibody against human beta-2-microglobulin.
abstract: Single domain antibody (sdAb) is often expressed as inclusion bodies in Escherichia coli cytoplasm. Establishing an effective in vitro refolding method for sdAb obtained from inclusion bodies would be important for sdAb research. In this study, dilution refolding condition for a camelid sdAb specific against human beta-2-microglobulin was optimized for the sdAb purified from the inclusion bodies of E. coli BL21 (DE3). Single factor methods based on protein concentration, velocity of dilution, incubation time and refolding buffer composition were first investigated. Then the key refolding buffer compositions were selected for further optimization by means of the Box-Behnken design of response surface methodology (RSM). The activity of the refolded sdAb was determined by measuring its specific antigen-binding ability using indirect ELISA. The optimized refolding condition of sdAb consisted of a 10-fold dilution in 10 mM Tris-HCl (pH 8.0) containing 1.24 mM GSH, 1mM GSSG, 352 mM L-Arg, 0.65% PEG-2000, and a 16 h incubation at 4 °C. Further comparison of the activities between the refolded sdAb and purified soluble sdAb expressed in E. coli Rosetta-gami (DE3) pLysS indicated that the sdAb was correctly refolded, as assayed by isothermal titration calorimetry. This work could provide an important strategy for the recombinant production and application of sdAb.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Bao
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Lu
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, China. Electronic address: lyj81@dlut.edu.cn.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Jia
mesh_terms: D000818:Animals; D002162:Camelus; D004926:Escherichia coli / Q000737:chemistry; D006801:Humans; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000302:isolation & purification; D001613:beta 2-Microglobulin
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D001613:beta 2-Microglobulin
keywords: Beta-2-microglobulin; Dilution refolding; Single domain antibody