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One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector
Qi Zhao1; Yin-Wah Chan1; Susanna Sau-Tuen Lee2; Wing-Tai Cheung1
2009
Source PublicationPROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
Volume68Issue:2Pages:190-195
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.

KeywordAntibody Hexahistidine Tag Immobilized Metal-affinity Chromatography Pcantab His Phagemid Vector Phage-displayed Antibody Library Scfv
DOIhttps://doi.org/10.1016/j.pep.2009.08.004
Indexed BySCI
Language英语
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS IDWOS:000272103000009
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Cited Times [WOS]:15   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionFaculty of Health Sciences
Corresponding AuthorWing-Tai Cheung
Affiliation1.School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
2.Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
Recommended Citation
GB/T 7714
Qi Zhao,Yin-Wah Chan,Susanna Sau-Tuen Lee,et al. One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector[J]. PROTEIN EXPRESSION AND PURIFICATION,2009,68(2):190-195.
APA Qi Zhao,Yin-Wah Chan,Susanna Sau-Tuen Lee,&Wing-Tai Cheung.(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),190-195.
MLA Qi Zhao,et al."One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector".PROTEIN EXPRESSION AND PURIFICATION 68.2(2009):190-195.
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