Castor RcDELLA(GAI) Protein Expression,Purification And Crystal Growth Conditions Filtering

Ricinus communis L.is an important oil crop with high economic value and market position.In recent years,dwarf castor has the advantages of high yield and mechanized harvesting,it has become the main direction of ramie research workers.Gibberellin(GA)is an important hormone regulating plant height,DELLA protein is a key regulatory element of GA signal transduction pathway,negatively regulates the GA signaling pathway and participates in the degradation of GA,which suppresses plant growth and development.This study intends to use the E.coli expression system to induce the expression of RcDELLA gene,the protein was purified by multi-step chromatography and screened for crystal growth conditions to lay the foundation for further studying biological functions and structural analysis of DELLA.In this study,RcDELLA(GAI)gene was cloned and seven prokaryotic expression vectors such as pHAT-2-RcDELLA were constructed.The gene heat shock transformation of E.coli BL21(DE3)expression strain and induced expression at 0.1mM IPTG,16℃,12-15 h,the obtained protein was purified by immobilized metal-ion affinity chromatography,ion exchange chromatography and gel filtration chromatogra-phy to obtain a protein with good chargeability and good monodispersity.The mass spectrometry showed that it was the target protein with a molecular weight of 62.853 KD.The protein was present in monomeric form.There are signs of crystal growth under three buffer conditions: 0.1 M Sodium citrate tribasic dihydrate pH 5.0,30%V/V Jeffamine ED-2001 pH 7.0,0.2 M Ammonium acetate,0.1 M Tris pH 8.0,16%W/V Polyethylene glycol 10000,0.2 M Sodium nitrate,20% W /V Polyethylene glycol 3350,the crystal properties need further verification.