Synthesis, Recombinant Expression and Functional Analysis of Filamentousphage Gene V Protein

Abstract

Abstract: Objective: To explore the gene synthesis, recombinant expression and functions of filamentousphage gene V protein (GVP). Methods: According to the gene sequence of GVP, eight oligonucleotide fragments with the selected E. coli-preferred codon were designed, and the GVP gene sequence was synthesized by overlap extension PCR. Then the synthesized sequence was inserted into pET-28a-c(+) plasmid. The recombinant plasmids obtained were transformed into E. coli to screen positive isolates of GVP. GVP expression in the positive strains was induced with IPTG. The recombinant proteins were purified by Ni+-NTA affinity chromatography. Results: GVP gene was successfully synthesized and highly expressed in E. coli BL21 (DE3) under the induction of IPTG, and the equilibrium dissociation constant was 7.27 × 10-5 mol/L between GVP and ssDNA. Conclusion: The recombinant GVP has high affinity with ssDNA and therefore can be used for the ssDNA detection of some pathogenic microorganisms in food.

Key words: gene V protein, E. coli BL21 (DE3), recombination, expression

CLC Number:

Da-Chuan ZHANG. Synthesis, Recombinant Expression and Functional Analysis of Filamentousphage Gene V Protein[J]. FOOD SCIENCE, 2012, 33(1): 191-194.

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