SUMO-Fused expression and characterization of Heparinase I from Bacteroides thetaiotaomicron

Abstract:

[Objective] To clone and recombinant express the gene Heparinase I from Bacteroides thetaiotaomicron, and then characterize the recombinant SUMO-Bt-HepI and Bt-HepI. [Methods] Codon optimization was done on the gene sequence of B. thetaiotaomicron heparinase I. The target gene was obtained by PCR amplification, inserted into the expression vectors pET-28a and pE-SUMO, and then transformed into E. coli Rosetta (DE3) to obtain recombinant products Bt-HepI and SUMO-Bt-HepI. Heparin sodium was used as substrate to study the enzymatic properties of the recombinant proteins. [Results] SDS-PAGE analysis showed that the molecular weights of Bt-HepI and SUMO-Bt-HepI were about 42.5 kDa and 55 kDa, respectively. Compared with Bt-HepI, the specific enzyme activity of Heparinase I increased by 48.9% after fusion SUMO-Tag. The enzymological properties showed that the optimum pH and temperature of Bt-HepI and SUMO-Bt-HepI were pH 9 and 45℃, and both recombinant enzymes were stable at pH 5-9, while the acid resistance of SUMO-Bt-HepI was obviously higher than Bt-HepI when the pH value was lower than 5. Besides, SUMO-Bt-HepI also showed higher activities than Bt-HepI under 50℃. In addition, Ca²⁺ and Mg²⁺ have obvious promoting effect on the recombinant heparinase I, while Cu²⁺, Mn²⁺ and Zn²⁺ show certain inhibiting effect, suggesting that in addition to the well-known Ca²⁺ binding site, Mg²⁺ binding sites may aslo exist in the structure of B. thetaiotaomicron Heparinase I. [Conclusion] Recombinant Heparinase I in B. thetaiotaomicron using SUMO fusion system significantly improved its specific enzyme activity for potential production and application.