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Abstract In this study, a multipurpose M13KE phage display vector was constructed from wild-type M13KE phage for long peptide or protein display libraries without helper phage to expand the scope of targeted high-throughput screening. Based on the relationship between the structure and function of minor coat protein of wild-type M13KE (wt-pIII), a truncated gene III (tgIII) encoding minor coat protein from M13KE phage was cloned. A fusion gene fragment harboring a lac/tac promoter, signal peptide and C-terminal region sequence of gIII was assembled with SOEing-PCR (splice-overlapping-extension polymerase chain reaction) method and inserted into M13KE vector. SDS-PAGE and Western blot analysis with anti-M13 pIII monoclonal antibody were employed to detect the expression of recombinant protein. c-Myc and HA tag sequences were fused into the recombinant protein. The results showed that tgIII was inserted into an unessential region of M13KE. According to the results of SDS-PAGE and Western blot with anti-M13 pIII antibody, pIII was expressed by wt-gIII and tgIII. gIII harboring two tags expressed both c-Myc and IIA peptides using SDS-PAGE and Western blot with the corresponding monoclonal antibodies. In this study, a multipurpose M13KE phage display system was successfully constructed, which could express both short and long peptide libraries without helper phage. In future, the obtained M13KE phage display system may be used for targeted high-throughput screening of long peptide libraries without helper phage.
Key words High-throughput screening; Phage display system; M13KE; Long peptide library
Phage display system is a laboratory biotechnique that fuses exogenous proteins or polypeptide expression products with coat proteins of filamentous bacteriophages to display the exogenous proteins or polypeptide expression products on their surface, thereby obtaining phages expressing specific proteins or polypeptides by screening for enrichment culture 1 , which directly links the genotype and phenotype to screen target proteins or polypeptides based on its specific affinity to ligands. M13KE, fd and fe are the commonly used filamentous bacteriophages in phage display system, which can infect E. coli carrying F' via gill, and most exogenous genes are inserted into gill and displayed in the form of fusion proteins on the surface of phages 2 .
However, excessively long exogenous genes will cause the loss of infection ability of phages. Therefore, the existing display systems are only capable of expressing...