The Influence Of Fusion His6-tags On ZmSR Activities And Crystallizations

Serine racemase(SR),a PLP-dependent enzyme,catalyzes D-serine and L-serine racemization and dehydration reactions.SRs from several plant species are overexpressed and characterized.We had overexpressed the C-terminally His6-tagged ZmSR in Escherichia coli.However,the fused tag and additional DTT on the enzymatic activities are not known.For this reason,we conducted some studies and the results are as follows:1.In this study,we constructed ZmSR with a fusion N-or C-terminal His6-tag,and detected the expression in Escherichia coli.The tobacco etch virus protease(TEVp)recognition sequence was incorporated between N-terminal His6-tag and ZmSR.2.The soluble expression and purification of recombination proteins were analyzed.After induction 24 h in 16℃ and 12 h in 28℃,proteins were soluble expressed by analyzes of SDS-PAGE.Recombination ZmSRs was purified by Ni-NTA and SDS-PAGE showed a single band.The yield of ZmSR was affected by the different positions of His6-tags.The N-terminal His6-tag was removed by TEVp,and non-tagged ZmSR was purified.3.The influence on promoting protein solubility of supplementary glycerol was analyzed.The supplementary 5% glycerol during protein preparation increased protein production and increased the ZmSR activities.His6-ZmSR was the most remarkably,because of its 28 times growth of production.The yield of ZmSR-His6 was more than,probably due to the incorporated TEVp recognition sequence.4.The protein properties of ZmSR was analyzed.When installing Superdex 200 Hiload 10/300 GL on FPLC,purified ZmSR-His6 displayed the sole peak at 280 nm and the protein molecular weight was 82.0 kDa calculating by protein marker.A subunit molecular mass was about 36 kDa by SDS-PAGE,representing that ZmSR is a homodimer in the solution.5.The activities of ZmSR was analyzed.The non-tagged ZmSR showed only higher activities of D-to L-Ser racemation and the other three activities was lower than His6-tagged ZmSR.His6-tag in diverse terminal differentiated the racemase activities and dehydratase activities.D-Ser racemase activities and dehydratase activities were His6-ZmSR higher than ZmSR-His6,L-Ser racemase activities and dehydratase activities were the opposite.6.The influences on ZmSR activities of reducing agent were examined.Increasing concentrations of DTT,the L-Ser racemase activity of non-tagged ZmSR was increased but D-Ser racemase activity was decreased.When DTT was more than 4 mM,the irritation and inhibition were both not that significant.In the presence of 1 mM DTT,L-Ser racemase activities racemase activities of ZmSRs were higher than D-Ser racemase activities,but L-Ser dehydratase activities were lower than D-Ser dehydratase activities.Futhermore,the four catalytic activities of C-terminally His6-tagged ZmSR were all higher than non-tagged ZmSR.7.Crystal growth of ZmSR.Microcrystals were grown in No.18、28、46、95 conditions of Hampton Research the company’s Crystal Screen 2-HR2-112 and No.70 condition of Crystal Screen 2-HR2-144.After further optimization screening,crystals in the No.46 condition of 98 kinds of screening kit(0.2 M calcium acetate hydrate,0.1 M Sodium cacodylate trihydrate pH6.5,18%(w/v)PEG8000)was obtained and a set of data was gathered.In conclusion,we prove that fusion tags are benefit to the soluble expression and purification of recombination protein.Fusion tags and reducing agent have influence on protein activity.These results suggest that fusion tags have advantage in screening recombination proteins.