Prokaryotic expression and purification of VWD domain in vitellogenin of Cherax quadricarinatus

【Objective】Prokaryotic expression of the vitellogenin(Vg) VWD domain of red claw crayfish(Cherax quadricarinatus) not only provided technical support for further research on the biological function of Vg and the development of corresponding bioactive substances, but also laid foundation for improving C. quadricarinatus culture, accelerating ripening and obtaining high-quality offspring.【Method】The protein sequence of VWD domain(2345-2491 aa) was found according to the Vg mRNA sequence (accession number:AF306784.1) of C. quadricarinatus published in GenBank database. Bioinformatics analysis and theoretical evaluation were carried out by using online softwares such as ExPASyProtParam, ProtScale, InterProscan and TMHMM. Optimization of original VWD amino acid sequence combined with codon preference of Escherichia coli. Then the target gene was obtained by whole gene synthesis, and the recombinant plasmid E. coli pET-28a-VWD was constructed by connecting to vector pET-28a, and transformed into positive clone conversion BL21 (λDE3) competent cells which were induced by IPTG. The induced fusion protein was detected by 10% SDSPAGE electrophoresis and Western blotting.【Result】The relative molecular weight of VWD domain of C. quadricarinatus was 19692.11 Da, theoretical isoelectric points(pI) was 9.35, and the molecular formula was C₈₅₅H₁₃₃₄N₂₅₈O₂₆₁S₉. It was an unstable hydrophilic protein and did not contain a transmembrane domain. Extended chain and irregular curl were the main elements of the secondary structure of VWD domain in C. quadricarinatus, among which, α-helix accounted for 7.73%, β-turn accounted for 10.50%, the extended chain accounted for 35.91%, and the irregular curl accounted for 45.86%. The recombinant plasmid pET-28a-VWD expressed the fusion protein VWD by inducing the competent cells E. coli BL21(λDE3). After dissolution with 2 mol/L guanidine hydrochloride and purification and renaturation on Ni-NTA affinity chromatography column, a clear specific band appeared at the relative molecular weight of the protein about 20.0 kD by 10% SDS-PAGE electrophoresis and Western blotting. The expression form of fusion protein VWD was mainly inclusion body. The best induction conditions for high expression of the fusion protein VWD in the competent cells BL21 (λDE3):when the OD_{600 nm} of the monoclonal bacterial solution reached 0.6, 0.5 mmol/L IPTG was added and cultured at 20℃ for 16 h. The concentration of purified fusion protein VWD was 1.32 mg/mL.【Conclusion】The optimized VWD gene of C. quadricarinatus obtained by whole gene synthesis can be expressed in E. coli competent cells BL21(λDE3), and the expression form of fusion protein VWD is mainly inclusion body. After dissolving with guanidine hydrochloride and purifying and renaturating with Ni-NTA affinity chromatography column, high-purity active VWD protein can be obtained, which provides technical support for the subsequent research on the biological function of C. quadricarinatus Vg and the development of corresponding bioactive substances.