Abstract: In order to perform isothermal titration calorimetry (ITC) experimentsin vitro, pXMJ19-gt1-His recombinant plasmid was constructed.Selected by single-factor optimization experiments, the optimal host cell for the glycerol transporterGT1expression was C41(DE3); the optimal expression condition was adding 0.01mmol/L Isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 0.7 and inducing at 30℃, 110r/min for 10h. It was identified by ultracentrifugation that some GT1 protein could be located to the plasma membrane of Escherichia coli. The detergent NP-40 with the highest resolving efficiency for GT1 was screened out. The GT1 protein was purified using nickel ion affinity chromatography with detergent buffer, and its glycerol binding affinity was measured by ITC. The results showed that the binding process was exothermic reaction, GT1 interacted with glycerol in vitro with a KDvalue of 6.58μmol/ L. The experiments showed that the GT1 protein was active and it could interact with glycerol in vitro, which provided a research basis for the study on crystal structureand function of GT1.

Key words: Pichia pastoris, glycerol transporter, membrane protein expression, major facilitator superfamily, protein-molecule interaction