One-step on-column purification and refolding of a single-chain variable fragment (scFv) antibody against tumour necrosis factor alpha.
Single-chain variable fragment (scFv) is a low-molecular-mass recombinant antibody and is usually expressed as inclusion bodies in Escherichia coli. Highly efficient purification and refolding methods are required to provide enough active proteins for therapeutic or diagnostic use. In the present study, an anti-TNFalpha (tumour necrosis factor alpha) scFv (TNF-scFv) was constructed and expressed in E. coli BL21(DE3) star as inclusion bodies, and a convenient procedure of one-step on-column purification and refolding was provided for it. Briefly, denatured TNF-scFv was firstly captured by immobilized metal (Ni) affinity chromatography, and then non-denaturing detergent (Triton X-100)-containing and beta-cyclodextrin-containing solutions were loaded in turn on to the column to perform 'artificial chaperone-assisted refolding' after removing impurities. More than 77% of denatured TNF-scFv protein was refolded successfully with a purity of more than 95%. Activity assays showed that refolded TNF-scFv could bind to rhTNFalpha (recombinant human TNFalpha) specifically with high affinity. It could inhibit rhTNFalpha from binding to TNF receptors and neutralize the cytolytic activity of rhTNFalpha against L929 cells effectively. A conclusion was obtained that this refolding method is time-saving and suitable for industrial production. It may also be applicable to other scFvs or other recombinant proteins.
Liu
Mengyuan
M
The Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Science, 100050 Beijing, People's Republic of China.
Wang
Xiangbin
X
Yin
Changcheng
C
Zhang
Zhong
Z
Lin
Qing
Q
Zhen
Yongsu
Y
Huang
Hualiang
H
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha