Modification Of Fusion Tag Pantothenyl Ethylamine And Its Recombinant Expression With Penicillin G Acylase

Acyl carrier protein(ACP)plays an important role in the synthesis of fatty acid metabolism in living bodies and has been extensively studied and utilized.The protein can from an inactive acyl carrier protein(apo-ACP)became an active acyl carrier protein(holo-ACP)with the catalysis of 4'-phosphopantetheine transferase,but the detective operation is complicated for this modification process.In this experiment,the4'-phosphopantetheine transferase(Sfp)from Bacillus subtilis was recombinantly expressed and purified,also optimized the detection system for ACP-specific modification.Meanwhile,the recombinant expression of ACP and penicillin G acylase(PGA)was explored,which has important application value of PGA.The highly active sulfhydryl group of holo-ACP can efficiently direct the orientation and covalent immobilization of proteins(enzymes),the result of study could provide reference for the orientation and covalent immobilization of PGA.The results of this study show that after the ACP is modified by Sfp,the4'-phosphopantetheine of CoA molecule can be efficiently transferred to apo-ACP with the catalysis of Sfp,so the specific modification is completed.The Ellman method is used to determine the CoA in the system.Changes in content can be used to quantitatively observe the specific modification process;this method is simple and can provide technical support for the in vitro modification of fusion proteins containing the Sfp recognition site(short peptide of SRP).The ACP and the recognition sites SRP of Sfp were respectively fused to the alpha subunit and beta subunit of penicillin G acylase,then recombinantly expressed in E.coli.The result of the fusion protein expression show that the proper concentration of the ?subunit and the ACP-? can be respectively purified,also the ACP-? can be obtained by digestion.The hydrolysis of penicillin G potassium salt showed some activity after the two fusion proteins were mixed in vitro.This experiment initially established a detection system for Sfp-mediated enzyme-specific modification,which provided a good technical support for the orientation and covalent immobilization of proteins(enzymes).The activity of PGA deserve further exploration by recombinant expression.