The Heterologous Expression Of A Dipeptidase Gene Of Inonotus Obliquus

Inonotus obliquus generally grows in cold regions,and Russia,Finland and other places all have their suitable growth environment.As a kind of fungi,its shape is also unique.In general,a large number of melanin products are accumulated on the surface of oroborus obliquus,some of which are granular,with the hinges of cell wall,and the other part of water solubility.Based on the analysis of Antismash,the water-soluble melanin of Betula NRPS-PKS is closely related to the gene cluster of non-ribosome-peptide,which contains not only the gene of genes,but also 10 genes such as AAA,HCP,DP and PK.The main research in this paper is DP gene,according to the analysis of the gene cluster,the DP gene has the peptide bond hydrolase structure domain,we speculate that it is a kind of dipeptide enzyme,may have the structure modification to the NRPS-PKS expression product,and the scientific research shows that the peptide enzyme and the two peptides synthesized by the reverse catalysis of two peptides are in the food,the application of medicine,feed and biology is more and more extensive,so it is of great scientific significance to study the function of DP in this gene cluster.Based on the above information,this paper mainly carried out the following researchIn this experiment,following studies were mainly developed:1: Use some online tool,such as NCBI,Prota Param,Signal P4.1,TMHMM2.0,and SWISS-MODEL to analyze the p Hysical and chemical properties of the DP gene,signal peptide,domain,and three-dimensional structure of transmembrane on the basis of bioinformatics,which guides for the next step.2: Respectively transform the both constructed recombinant plasmid TDP-p PICZ?A with a transmembrane region DP gene,and DP gene-free transmembrane region DP-p PICZ?A into Pichia pastoris x-33,to investigate whether transmembrane region has an impact on protein expression.3: Lactococcus lactis shows evident advantages in expression of membrane proteins.The recombinant plasmid DP-PNZ8148 with transmembrane domain DP gene is constructed and expressed using the Lactococcus lactis NICE system.4: Purify DP-free recombinant protein expressed by Pichia pastoris,preliminarily verify its function,and deplore its enzymatic properties of the dipeptide substrate Gly-Gly.The results estimate that the coding region of the DP gene is 1395 bp in length with 465 amino acids,which presents the highest similarity to membrane dipeptidase.Meanwhile it has a transmembrane domain while no signal peptide,which can further demonstrate that DP is a membrane protein,not secreted protein;DP gene recombinant plasmid with transmembrane region and non-transmembrane region are transformed into Pichia pastoris.It is found that transmembrane region DP fail to express in Pichia pastoris,while DP without transmembrane region can realize the desirable result,which elaborating that there are certain limitations in the Pichia pastoris expression of membrane proteins.We successfully expressed the whole-length DP genen through the NICE system of Lactococcus lactis NICE in order to investigate whether the presence of transmembrane regions has an effect on the function of DP membrane proteins.The whole-length DP gene was explored in subsequent experiments.Finally,purify the recombinant protein without transmembrane domain expressed by Pichia pastoris,and preliminarily verify the function of the DP membrane protein.It was found that peptidase is equipped with excellent properties,and it shows better activity when the substrate was Gly-Gly.The specificity of the dipeptidase is for further study.