Cloning, Expression, Purification And Characterization Of A 2-ketogluconate Metabolic Regulation Protein—PtxS From Pseudomonas Plecoglossicida JUIM01

2-ketogluconate(2KGA) is an important organic acid and mainly used as the key intermediate for producing erythorbic acid(isoascorbic acid). This study aimed to clone2-ketogluconate metabolic regulatory protein Ptx S from an industrial 2KGA producer of Pseudomonas plecoglossicida JUIM01 and the gene was expressed and the product was isolated and purified.Based on our previous research, the present study characterized the structure and functions of Ptx S by using Bioinformatic analysis, Gene Knockout, SDS-PAGE,MALDI-TOF-MS, Circular Dichroism, Dynamic Light Scattering, and Isothermal Titration Calorimetry. The obtained results will provide a theoretical basis for constructing the 2KGA efficient producers further.The obtained results and conclusions are listed as follows:(1) The Ptx S gene for the first time was amplified by TD-PCR. The cloned ptx S had a complete open reading frame(ORF) of 1023 bp encoding 340 amino acids. The amino acid sequences of JUIM01-Ptx S had the sequence identities of 88%, 84% and 74% with those of P.putida, P. mosselii and P. plecoglossicida Ptx S, respectively. The amino acid sequences alignment result showed that four Ptx S protein DNA binding domains located at the N terminus with a high homology and contained a helix-turn-helix motif. Ptx S had the theoretical molecular weight of 36651.7 u and p I value of 6.39. Ptx S was situated in the cytoplasm, without transmembrane domain and signal peptide, and belonged to a hydrophilic protein. The protein contained a variety of active sites, such as protein kinase C phosphorylation sites. The predicted secondary Ptx S structure contained 54.41% of α helixes,13.53% of extended strand, 10.88% of β turns and 21.18% of random coil. Protein interaction network results showed that Ptx S protein probably was close to some enzymes involving in2 KGA metabolism and existed at the same time.(2) The expression plasmid p ET-28a-ptx S was constructed by cleaving the Ptx S gene fragment and the p ET-28 a with Nde I and Xho I and ligating with a DNA ligation kit and the recombinant strains E. coli BL21(DE3)/p ET-28a-ptx S was constructed. Polyacrylamide gel electrophoresis showed that recombinant expression strains had higher expression levels in the vicinity of 36.7 ku, which was slightly higher that predicted by using Prot Param(http://web.expasy.org/protscale/) software. Ptx S was extracted and purified by using ultrasonication, centrifugation, freezing precipitation, Nickel-affinity and gel filtration chromatography. CD spectrum showed that Ptx S contained 52.3% of α helixes, 13.53% of extended strand, 10.88% of β turns and 21.18% of random coil, which is similar to the results predicted with SOPMA. Dynamic light scattering indicated that the purified Ptx S protein was a monomer with particle size, molecular weight and polydispersity index(Pd) of 2.554530 nm,30 ku and 23.2%, respectively.(3) According to the biological information of ptx S, the ptx S in-frame deletion fragment was cloned by overlap-PCR, and the ptx S deletion mutant Ps. plecoglossicida JUIM01Δ ptx S was constructed. It could be found that Ptx S repressed the 2KGA metabolism by comparing the 2KGA fermentation profiles of Ps. plecoglossicida JUIM01 and JUIM01Δ ptx S.Isothermal titration calorimetries(ITC) showed that 2KGA could specifically bind to Ptx S in vitro and lead to the secondary structural changes of Ptx S protein. Hence, it could be concluded that 2KGA is an effector molecule of Ptx S protein. By comparing the2-ketogluconate utilization operon(kgu operon), the 14 bp palindrome was situated at the5’ end of kgu operon gene. ITC analysis also showed that the purified Ptx S protein could specifically bind to palindrome(5’-TGAAACCGGTTTCA-3’) in vitro.