Gene Synthesis, Expression And Property Research Of Protein-glutaminase

Most plant proteins have poor solubility and functionality, because of the contents of glutamine residue are generally high, resulting in their limited use in foods.Deamidation of proteins can improve protein functionalities such as solubility, emulsification, and foaming and gelation properties, which are desired properties in some food proteins. Therefor, deamidation of such proteins is one of the most promising ways to expand their uses and to improve their functionalities. Protein deamidation (PG), was discovered in culture supernatants of Chryseobacterium proteolyticum,which hydrolyses the amido groups on glutamine or asparagine residues within proteins, attracts a great deal of attention in the food industry, as this reaction has potential applications as a method for improving the usefulness of a variety of food proteins. This research expressed protein glutaminase via means of genetic engineering, provided an important reference for the research and development of PG enzyme.The main content of the research including the following three aspects:1. Artificial synthesis of the protein glutaminase gene.The PG full length gene sequence was optimized and designed into24overlapping oligonucleotide fragments, each fragment is of about60bp, and the overlapping portion is about20bp. These24oligonucleotides were spliced into a full-length PG gene by SOE-PCR. The PG gene was cloned into pUC19-T vector for sequence analysis, the result show that the long fragment gene occurred frameshift mutation because of the position of the724bp inserted a thymine (T). The full-length gene was site-directed mutagenesis by overlap extension PCR, and the PCR products were sequenced to obtain the correct sequence.2. Expression and Property Research of Protein-glutaminase in Escherichia coli expression system.The gene encoding the protein was synthesized using overlap extension PCR and the mature PG gene was cloned into expression vector pET32a(+).The recombinant protein was expressed in Escherichia coli BL21(DE3)as inclusion bodies via IPTG induction and lactose self-induced pathway, the resluts show that the yield of recombinant protein by lactose-inducible expression pathway is5.3times than the IPTG induction. In order to improve the solubility of PG, the culture was incubated in cold-shocked condition and a chaperone plasmid pTf16-tig was also cloned into pET32a-matPG/BL21(DE3). The results showed that low temperature can improve the solubility of PG slightly,but pTf16-tig was futile. The active PG was obtained after denaturation and renaturation,then the PG was purified by Ni affinity chromatography.For deamidating activity assay, Cbz-Gln-Gly was used as substrate. The reaction showed that PG can effectively hydrolyze glutaminyl residues in the Cbz-Gln-Gly and resulting in the release of ammonia. The research on enzymatic properties of PG showed that the optimum temperature is40℃and the optimal pH is6.0.3. Expression and Property Research of Protein-glutaminase in Bacillus subtilis expression systemIn this issue, we build two new Escherichia coli-Bacillus subtilis shuttle vector pHPW and pCW.To construct pHPW, the fragment p43-nprB including strong promoter p43and neutral protease signal peptide nprB were seamlessly connected by overlap extension PCR,then amplicon was cloned into pHP13to obtain the shuttle vector pHPW.Production of recombinant proteins at low temperatures is one strategy to prevent the degradation due to their own protease. We constructed a novel cold-inducible vector pCW,which contains the cold-inducible des promoter,and the coding region for the signal peptide of the SamyQ gene encoding an a-amylase fused to the des promoter.The mature PG gene was cloned into shuttle vector pHPW and pCW resulting recombinant plasmid pHPW-PG and pCW-PG. Next, the recombinant plasmid was transformed into B. subtilis DB403. The expression levels of PG was detected by SDS-PAGE, and the results show that shuttle vector pHPW and pCW could secreted PG enzyme out of the cell efficiently. We purified PG enzyme from the culture supernatant of pHPW-PG/DB403and pCW-PG/DB403. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. The reaction showed that PG can effectively hydrolyze glutaminyl residues in the Cbz-Gln-Gly and results in release of ammonia. Enzymatic casein experiments showed crude PG enzyme can improve the solubility of the casein.In summary, the two new high expression vectors pHPW and cold-inducible expression systems pCW provide an inexpensive alternative technology especially for industrial production of recombinant proteins. The two genetic engineering bacteria pHPW-PG/DB403and the pCW-PG/DB403constrcted in this research can express active PG, these data provide an important reference for future research and development of Protein-glutaminase.