Expression,Purification,Crystal Growth And Functional Analysis Of RcRBCL,a Ribose-1,5-Bisphosphate Carboxylase/Oxygenase From Ricinus Communis

Ribose-1,5-diphosphate carboxylation / oxygenase(Rubisco)exists in all photosynthetic organisms,which plays an important regulatory role in photosynthesis and is a key enzyme for photosynthetic carbon assimilation.Rubisco is a limiting factor of photosynthetic rate and an important target factor for improving crop photosynthetic efficiency.Rubisco is composed of eight small subunit(RBCS)and eight large subunit(RBCL),and RBCL mainly plays a catalytic role.By cloning Rc RBCL gene,prokaryotic expression and purification system was established,and crystal growth conditions were screened to obtain protein crystals.Yeast two-hybrid(Y2H)technology was used to verify the interaction between Rc RBCL protein and Rc GST-F11 protein point-to-point,aiming to provide certain experimental basis for the function of Rc RBCL protein and its molecular mechanism in the photosynthetic pathway of castor.A series of prokaryotic recombinant protein expression vectors were constructed such as pETM13-RcRBCL and pETM50-RcRBCL,and the recombinant protein expression was induced by IPTG with E.coli BL21(DE3)as the host.When the induction conditions were 0.1 m M IPTG,incubated at 37 °C and 220 rpm for 12 h,the recombinant proteins were insoluble.The solubility of Rc RBCL recombinant protein was increased by renaturation.It was found that the solubility of Rc RBCL recombinant protein was higher when 8 M urea(p H 8.0)solution was used as denaturant,20 m M Tris-HCl,p H 8.0 and 500 m M Na Cl solution was used as renaturation buffer.The recombinant protein was further purified by affinity chromatography,anion exchange chromatography and molecular sieve chromatography.It was found that the recombinant protein existed in the form of polymerization and was tolerant to low salt.Finally,the homogeneous and high purity recombinant protein was successfully obtained.The crystal growth test of RcRBCL-His protein was carried out using the crystal growth screening kit.When the liquid volume in the crystal growth cell was 100 μL,the protein concentration was 20.23 mg / m L,the sample volume was 2 μL,the ratio of cell liquid to protein was 50 % : 50 % and 25 % : 75 %,respectively,the precipitant was20 % PEG 3350,and the buffer was 0.2M diammonium phosphate(p H 8.0),Rc RBCL-His produced crystals in the sample cell that were different from the salt crystal morphology in the cell liquid,showing microcrystalline and polycrystalline crystals.The analysis of interaction results showed that blue plaque was clearly observed on QDO / X / A(SD /-Leu /-Trp /-His /-Ade / X-α-Gal / Ab A),indicating that there was a direct interaction between RcRBCL and RcGST-F11.It is speculated that Rc RBCL may be regulated by Rc GST-F11,which affects the sugar metabolism pathway by regulating the carbon fixation process of photosynthesis in castor,thereby regulating plant growth and development.The above results provide a new perspective and experimental basis for the elucidation of Rc RBCL protein function and its molecular mechanism.