Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins

¹ College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.; Key Laboratory for Supramolecular Structure and Materials, Jilin University, Changchun, 130023, China.
² College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.; Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China.
³ College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.; The Third Clinical College, Jilin University, Changchun, 130031, China.
⁴ College of Pharmaceutical Science, Jilin University, 1266 Fujin Road, Changchun, 130021, China.
⁵ Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, China.
⁶ The Third Clinical College, Jilin University, Changchun, 130031, China.
⁷ College of Life Science, Jilin University, Changchun, 130023, China.
⁸ Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, 130023, China.
⁹ Biochemistry and Cell Biology Department, State University of New York at Stony Brook, USA.
¹⁰ Key Laboratory for Supramolecular Structure and Materials, Jilin University, Changchun, 130023, China.

^* To whom correspondence should be addressed. E-mail: jingyanwei{at}yahoo.com.cn.

	Abstract

A novel gene, testes-specific protease 50 (TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.

Key Words: antibody library, phage display, single-chain variable fragments (scFvs), testes-specific protease 50 (TSP50)

First published on April 28, 2006, doi:10.1177/1087057106287901

Journal of Biomolecular Screening 2006;11:546.

A more recent version of this article appeared on August 1, 2006


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