Protein Expression And Purification Of KCS6/CER2 Complex Of Plant Fae Component

Plant very-long-chain fatty acids（VLCFA）play an important regulatory role in plant growth and development.Derivatives are important sources of food,synthetic drugs,chemicals,petroleum and other necessities.The synthesis of VLCFA in plants has two stages:（1）synthesis of long-chain fatty acids catalyzed by fatty acid synthase（FAS）;（2）The formation of VLCFA from aliphatic chains catalyzed by Fatty acid elongase（FAE）.3-ketoacyl-Co A synthase（KCS）is a rate-limiting enzyme that catalyzes the first condensation reaction of long-chain fatty acids from plants to VLCFA.At present,KCS specific catalytic synthesis of VLCFA with different chain lengths has been found in yeast and plants,but the carbon chain length of fatty acids is less than 28,and the carbon chain length of fatty acids extends to more than 28 with the assistance of CER2（eceriferum2）.The mechanism of catalytic carbon chain extension by KCS and KCS/CER2 complex is not clear.In 2008,some scholars tried to explain the mechanism from the perspective of structural biology.However,as KCS protein is a membrane protein,it is very difficult to obtain stable and active KCS and KCS/CER2 complex.In this study,13 KCS family genes from Arabidopsis thaliana,WSL4 gene from rice,and 5 KCS genes with high expression at m RNA level from citrus were selected,and the interaction between CsKCS6 and Cs CER2 in citrus was tested and verified by cross-expression,and CsKCS6 and Cs CER2 complexes were expressed and purified.The following results were obtained:（1）The basic information,transmembrane region,conservative domain and tertiary structure of KCS/Cs CER2 protein were predicted according to relevant websites,and it was found that CsKCS6 was flexible in the 350-390 region of its amino acid sequence except that the transmembrane region 20-42 and 63-82 were unstable.The active HXXXDG sequence,H166 and H38 of Cs CER2 were surrounded by a large number of loop regions.（2）19 KCS homologous genes were fished and Cs CER2 protein was successfully expressed and purified in E.coli.Most of the KCS homologous protein samples expressed in E.coli existed in the form of inclusion body,and a small part of the target protein was not bound to affinity chromatography column.（3）In view of inclusion body phenomenon,the target protein was expressed in E.coli by fusion tag,expressing strain,protein truncation and other optimization methods.It was found that most of the protein samples still existed in the form of inclusion body,and a small part of the target protein was not bound to affinity chromatography column.（4）In view of the non-binding affinity chromatography column phenomenon of the target protein,CsKCS6^{78-466（（?）368-384+12GS）}protein can be purified by 20%ammonium sulfate by increasing His tag position and purifying the target protein by ammonium sulfate precipitation method,but the protein is precipitated and unstable.（5）CsKCS6 protein was optimized in Saccharomyces cerevisiae,insect cells,citrus suspension cells and mammalian cells.Finally,CsKCS6 protein was expressed and purified in mammalian cells.（6）The interaction between CsKCS6 and Cs CER2 was verified by pull-down technology,and the CsKCS6/Cs CER2 complex was expressed and purified by co-expression.The results of this study provide a basis for the structural analysis of CsKCS6protein and CsKCS6/Cs CER2 complex,provide a reference for the preparation of protein samples by KCS protease activity determination and other biochemical experiments,and further expand the idea of exploring the catalytic mechanism of carbon chain extension by KCS and KCS/CER2 complex.