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One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.
Protein Expr Purif 2009; 68(2):190-5PE

Abstract

Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

Authors+Show Affiliations

School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19683057

Citation

Zhao, Qi, et al. "One-step Expression and Purification of Single-chain Variable Antibody Fragment Using an Improved Hexahistidine Tag Phagemid Vector." Protein Expression and Purification, vol. 68, no. 2, 2009, pp. 190-5.
Zhao Q, Chan YW, Lee SS, et al. One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector. Protein Expr Purif. 2009;68(2):190-5.
Zhao, Q., Chan, Y. W., Lee, S. S., & Cheung, W. T. (2009). One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector. Protein Expression and Purification, 68(2), pp. 190-5. doi:10.1016/j.pep.2009.08.004.
Zhao Q, et al. One-step Expression and Purification of Single-chain Variable Antibody Fragment Using an Improved Hexahistidine Tag Phagemid Vector. Protein Expr Purif. 2009;68(2):190-5. PubMed PMID: 19683057.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector. AU - Zhao,Qi, AU - Chan,Yin-Wah, AU - Lee,Susanna Sau-Tuen, AU - Cheung,Wing-Tai, Y1 - 2009/08/13/ PY - 2009/05/14/received PY - 2009/07/23/revised PY - 2009/08/10/accepted PY - 2009/8/18/entrez PY - 2009/8/18/pubmed PY - 2010/1/23/medline SP - 190 EP - 5 JF - Protein expression and purification JO - Protein Expr. Purif. VL - 68 IS - 2 N2 - Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening. SN - 1096-0279 UR - https://www.unboundmedicine.com/medline/citation/19683057/One_step_expression_and_purification_of_single_chain_variable_antibody_fragment_using_an_improved_hexahistidine_tag_phagemid_vector_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(09)00202-2 DB - PRIME DP - Unbound Medicine ER -