Cloning, Expression And Function Of Protein G Igg Fc Binding Domain

Protein G IgG Fc binding domain (PGFB) was cloned and expressed, it was used to purify antibody. According to the amino acid sequence of PGFB, four oligonucleotide fragments were designed with E.coli-preferred codon. Then the gene of PGFB was synthesized by overlap extension PCR. After sequencing was finished, it was cloned into plasmid pET-28a(+). The recombinant plasmid was transformed into E.coli BL21(DE3) by calcium chloride transformation method. Recombinant protein was induced by IPTG and expression conditions were optimized. With the tag of 6×His in the plasmid pET-28a(+), the recombinant protein was purified by a single step affinity chromatography with Ni⁺-NTA. After purification was finished, the recombinant protein was coupled with sepharose 6B to purify polyclonal antibody.The result showed that the gene of PGFB was synthesized successfully. The recombinant plasmid pET-28a(+)-PGFB was constructed and the recombinant protein was expressed in E.coli BL21(DE3). When the IPTG concentration was 1.0 mmol/L and induction time was 9 hours, PGFB was expressed highly. After PGFB was purified, its purity reached 90% and its molecular weight was the expected 12.25 kilo Da. It was confirmed that PGFB had the ability to combine with polyclonal antibody IgG by Western Blot. Then the saturation curve was drawn and the maximum binding capacity of PGFB was (0.79±0.051)mol IgG/mol PGFB. According to Scatchard plot, the dissociation constant was (7.33±2.17)×10^-6mol/L. When the NaOH concentration was 1.0mol/L, the amount of epichlorohydrin was 2.5mL/g sepharose 6B and reaction time was 4 hours, sepharose 6B had the highest activation. PGFB was coupled with sepharose 6B, the product can purify polyclonal antibody. The binding capacity reached 20 mg IgG/mL medium.In this study, the recombinant plasmid pET-28a(+)-PGFB was constructed and high expression of PGFB provided convenience for the rapid purification of polyclonal antibody.