Study On Heterologous Expression Of Erythromycin Biosynthetic Proteins

Erythromycin is a polyketide synthesized by type I polyketide synthase.Polyketides have structural and biological activity diversity,so they are one of the important sources of new drug development.Erythromycin has a certain inhibitory effect on Gram-positive bacteria and Gram-negative bacteria.It has a broad-spectrum antibacterial activity and is often used to treat skin and soft tissue infections,conjunctivitis,pharyngitis and pneumonia.Series of erythromycin derivatives by modifying the structure of erythromycin have been produced.These derivatives have stronger antibacterial activity,lower toxicity and resistance to drug resistance than erythromycin.Study the biosynthesis mechanism of erythromycin,provide a theoretical basis for designing and producing more new polyketides,and discovering more effective drugs for the treatment of human diseases.DEBS is a polyketide synthase that contains three subunit proteins,and each subunit contains two modules that catalyzes the biosynthesis of erythromycin.At present,the heterologous expression of DEBS protein subunits in E.coli is relatively low,and the fine structure of partially truncated domains has been reported.The overall structure of modules,subunits and full complexes has not yet been reported.This study aimed at the problem of low yield of large heterologously expressed proteins.Taking the erythromycin synthesis machine as the research goal,the heterologous expression level of DEBS2 subunit was improved,and the in vitro expression and purification system of DEBS3 subunit was established.The high-purity DEBS3 protein with a molecular weight of 320 k Da was purified;it is proved that optimizing the N-terminal initial sequence of the target gene had a significant effect on the increase in the expression of large-molecular-weight DEBS2(374 k Da)protein.We made a preliminary observation on the structure of DEBS3 protein.For the low expression level of DEBS2,select 11 amino acids 33 bp in the initial region of the coding region of the gene.Introduce a 33 bp degenerate sequence at the beginning of the target gene by PCR.Random optimization to change codons,and then construct the expression vector using the PCR product,successfully selected the degenerate plasmid that highly expressed the target protein DEBS2 relative to the original plasmid,so that the expression of DEBS2 protein was improved.For the low expression level of DEBS3,construct expression vector of DEBS3 with n Flagc His tag.Optimize the expression conditions of DEBS3 protein: molecular chaperone co-expression,inducer concentration screening and medium screening.Finally the optimal expression conditions of DEBS3 protein were obtained:in TB medium,E.coli BAP1 was used as the host bacteria,and the molecular chaperone pGro7 plasmid was co-expressed,and the final concentration of100μM/L IPTG induced protein overexpression.At last,the soluble high expression of DEBS3 protein was obtained.DEBS3 protein was purified by Ni column affinity chromatography,Q column ion exchange,Flag column affinity chromatography and gel filtration chromatography.The protein was effectively separated and purified to obtain high-purity DEBS3 protein.The structure of DEBS3 protein was observed by negative staining electron microscope.The buffer of DEBS3 protein was optimized,and protein particles observable by electron microscope were initially obtained.There is no report on the structure of erythromycin single-module protein,so this study also successfully constructed single-module M2,M3,M4,M5 and M6 expression vectors,and obtained high-purity M3 protein.In this study,by optimizing the start sequence of the target gene,an E.coli strain with high expression of DEBS2 protein was successfully obtained,so that the expression level of DEBS2 protein in E.coli was from almost invisible on SDS-PAGE map to be purified;the in vitro expression and purification system of the giant erythromycin synthase protein subunit was established,and DEBS3 protein was explored negative staining structure.The high-purity large protein DEBS3 expressed in E.coli was successfully obtained and its structure was explored.It will lay the foundation for the next step to analyze the structure of DEBS2 and DEBS3 protein and erythromycin synthase.And provides a theoretical basis for the targeted transformation of the structure of erythromycin through genetic engineering or protein engineering.