The Research Of Glutamine Aminoacyl-tRNA Synthetase Complex Solubility And The Effect Of Regenerated Silk Protein On HRP

Aminoacyl-tRNA synthetase(AARS)is the key enzyme of catalyzing protein synthesis which is responsible for connecting amino acid to the homologous aminoacyl tRNA.In higher eukaryotes,AARSs not only have classic catalytic function,but also have many known and unknown nonclassic function.Glutamine aminoacyl tRNA-synthetase(QRS)is a kind of AARSs.There are few researches about the structure and function of human QRS.Because it is hard to get soluble and active human QRS protein,what's more,it is important to make QRS soluble.Therefore,this article introduce the methods about coexpression of QRS with its auxiliary proteins in E.coli and expression of QRS in pichia to get soluble human QRS protein.This article constructed two prokaryotic expression vectors Duet-RSS-N(1-67)-P43-N(1-37)and Duet-RSS-p43 in E.coli in order to coexpress with pET28a-QRS.According to the results,the solubility of QRS can be improved by coexpressing with Duet-RSS-N(1-67)-P43-N(1-37),compared with the independent expression of QRS.However,it can not get much soluble QRS protein.The coexpression of Duet-RSS-P43 and pET28a-QRS can get lots of soluble QRS protein complex.However,a large amount of QRS protein precipitate after the Ni column purification process.And there is not enough protein for crystal structure analysis in the supernatant fluid.In order to get enough soluble QRS protein,this article constructed eukaryotic expression vector pPICZ?-QRS in pichia to try to get soluble QRS protein.Comparing with the independent expression of pET28a-QRS in E.coli,QRS protein expressed in pichia is soluble but insoluble in E.coli.However,the amount of dissolved QRS protein is not enough for crystal structure analysis either.The results of this study can lay the foundation for the analysis of soluble QRS protein.This article also studied the regenerated silk fibroin products application in the direction of slow release behavior.After degumming,dissolved,dialysis and centrifugal,regenerated silk fibroin solution can be made.Fibroin membrane can form through natural drying,and silk fibroin hydrogel can be made by self-assembly.This article loaded horseradish peroxidase(HRP)onto the silk protein membrane which become water insoluble after the treatment of ethanol to research the protective effects by silk fibroin membrane on HRP enzyme activity and the slow release behavior.According to the results,regenerated silk protein membrane has no protective effect on the HRP protein about the thermal stability of HRP,but it has a certain slow release effect on HRP.In addition,HRP protein loaded into different concentrations of hydrogel prepared by self-assembly shows that 2% of silk protein hydrogel has better protective and slow-release effect on HRP protein compared with 1% and 0.5% silk protein hydrogel.There is not too much difference in the secondary structure of the three kinds of hydrogel by Fourier infrared detection.This experiment successfully loads the enzyme onto the silk protein membrane and into the silk protein hydrogel.This article can provide a reference for studies about the protection and slow release behavior of enzyme.