Construction Of Le-EIN3 Expression Vectors And Purification Of The Recombinant Protein

Ethylene is a very important plant hormone in tomato fruit growing and ripening, and Le-EIN3 is a downstream response factor. The expression of Le-EIN3 can activate many ethylene-response reactions as we known. EIN3 is positive regulator in ethylene-response pathway. As a important downstream response factor ,the function and mechanism of le-eil3 is not very clear. It's necessary for us to clone and express the tomato EIN3,if we want to make thorough researches on tomato fruit growing and ripening.The ACCESSION number of tomato EIN3 gene in GenBank is AF328786. The length of EIN3 coding sequence is 1844 bp. Primary sequence analysis indicated that it exhibited 79% sequence similarity to tobacco TEIL and the sequence similarity of this clone to A.thaliana EIN3,EIL1,EIL2 and EIL3 was 59%,56%,52% and 46% respectively. According to the sequence of EIN3 registered in GenBank,we designed primers and cloned the gene.After enzyme cutting ,ligation , transformation, we constructed two kinds of procaryotic express vectors(pET15b-EIN3,pET30a-EIN3) and one kind of eukaryotic express vector(pPIC9k-EIN3).The positive clones were analysed by PCR ,enzyme cutting and DNA sequencing. The pET15b-EIN3,pET30a-EIN3 were transformed into E.coli BL21 and pPIC9k-EIN3 was transformed into yeast KM71.After IPTG inducing in pET15b-EIN3/BL21,pET30a-EIN3/BL21 and methanol inducing in pPIC9k-EIN3/KM71 , the expression of exogenous protein was analysed by SDS-PAGE gel electrophoresis and ELISA. We compared the expression of the interest protein in the three kinds of engineering strains , then , we found the expression of EIN3 protein is more efficient in eukaryotic express strains than in procaryotic express strains. Four Pichia transformants that possesses the high yield of EIN3 protein were found after G418 multicopy screening and SDS-PAGE gel electrophoresis analysis. Then , the culture condition was optimized. Puryfied with Ni-chelating affinity chromatography,the interesting protein was attained.The purified protein was analysed by SDS-PAGE gel electrophoresis and ELISA. A reactive band,whose apparent molecular weight was about 71 kD. These results indicate that the recombinant tomato EIN3 protein was successfully expressed and purified.