Construction Of Serial New Typical Fusion Expression Vectors Of 5-Aminolevulinic Acid Synthase, Expressionand Purification Of Its Product In Escherichia Coli

5-aminolevulinic acid synthase(5-Aminolevulinic acid.ALAS)exists in all organisms,which is the key enzyme of heme synthesis in C4 pathway and catalyze succinyl-coenzyme A and glycine synthesizing ALA,CoA and CO₂.ALA is metabolic intermediates synthesizing heme,chlorophyll,vitamin B₁₂and tetrapyr,which has broaded application in agricultural production.It is not only used as herbicides,insecticides and also to increase the salt-resistant and anti-cooling capacity of plants,that is of great practical significance to ease the deterioration of the ecological environment and raise the level of agricultural production.In addition,5-aminolevulinic acid has been widely used for diagnosing cancer and treating cancer.Recently,it is difficult to obtain large quantities of natural ALAS,which blocks the research on structure and function.We construct three new typical ALAS fusion expression vector(pET-HMA.pET-HGA,pET-HDA)to gain natural protein..Three vectors were transferred into BL21 to induce and express,after one step purification to obtain high purity ALAS recombinant protein.The results showed:1 The primer and anti-sense primer were designed deliberately based on the published sequence of hem1 gene from Saccharomyces cerevisiae,which encodes the assumed mature ALAS from the first amino acid residue Asn63.The PCR reaction was performed including the designed primers,and Saccharomyces cerevisiae genome as the template. A main band about 1500bp was shown by agarose gel electrophoresis.The fragment was determined by sequencing.2.three new typical ALAS fusion expression vector(pET-HMA.pET-HGA,pET-HDA) were successfully constructed:those fusion proteins have 6 histidines and 3 solubility tags (GST,MBP,DHFR)which commonly increase the soluble expression at N-terminal,and the fusion protein has the TEV protease cleavage sequence.3.The three fusion expression vector of pET-HMA,pET-HGA and pET-HDA were transferred into BL21 and then induced to express.It showed that the three fusion proteins of MBP/ALAS,GST/ALAS and DHFR/ALAS by SDS-PAGE analysis were soluble expression proteins and the expression mass significantly increased.We did not gain whole fusion protein when MBP/ALAS was expressed.It fractured between ALAS and MBP in cell,Possibly because the peptide chain connecting MBP and ALAS was overlength that exposed to the outside of fusion protein so that it was hydrolyzed by protein enzyme in cell.That expressed fusion protein of GST/ALAS and DHFR/ALAS were gained,the molecular weight of which are 70kD.It Showed that GST and DHFR protein can enhance the ALAS correct folding and increase their soluble expression.4 The fusion protein DHFR/ALAS and GST/ALAS with His-Tag was one-step purified by nickel column.After SDS-PAGE analysis and purification,each recombinant protein was one band.It showed that using His-Tag to screen and purify aimed protein is a fast and effective method.In summary,encoding mature 5-aminolevulinic acid synthase gene fragment has been successfully cloned from Saccharomyces cerevisiae,and construct three efficient fusion expression vector in prokaryotic:pET-HMA,pET-HGA and pET-HDA.The efficient recombinant expression protein of ALAS was obtained in E.coli and was purified to homogeneity that provided a basis for studing crystal structure analysis,enzymatic mechanism and the function of ALAS.Meanwhile,the three prokaryotic efficient expression vector of pET-HMA,pET-HGA and pET-HDA provided a platform for efficient protein expression in prokaryotic system.