Enzymatic Synthesis Of N-acetyl-d-neuraminic And Positive Selection Expression Vector

N-Acetyl-D-neuraminic acid (Neu5Ac) is the key intermediate to chemical synthesis of Zanamivir, one of the two medicines used in the treatment of influenza. N-acetyl-D-neuraminic acid is currently made through enzymatic synthesis; the procedure consists of two steps:first, N-acetyl-D-glucosamine 2-epimerase (AGE) catalyzes the transformation of N-acetyl-D-glucosamine (GlcNAc) into N-acetyl-D-mannosamine (ManNAc), after which two pathways exist. One is the condensation of ManNAc and pyruvate into Neu5Ac through aldolase, and another is the condensation of ManNAc and phosphoenolpyruvate through synthetase.Through homology search, we found several proteins that have identity with N-acetyl-D-neuraminic acid synthetase (neuB) from E. coli K1. These proteins were cloned, expressed and purified. The activities of aldolase, NeuB and AGE from different sources were determined. The results show that E. coli aldolase has the best activity:after 1 hour incubation at 37℃, the yield of Neu5Ac from ManNAc was 23.31% in whole-cell catalysis. NeuB from Neisseria meningitidis showed the best synthetase activity, with a 50.96% molar conversion after 30 minutes incubation at 37℃in whole-cell catalysis. BT0453 from Bacteroides thetaiotaomicron VPI-5482 showed the best AGE activity in that when coupled with E. coli aldolase in whole-cell catalysis,2.77% molar conversion from GlcNAc was observed after 4 hours incubation at 37℃in whole-cell catalysis.Three E. coli chaperones were used as fusion tag to contruct chaperone-fusion protein expression vectors. Two proteins, A113695 and RB3348, were expressed in these vectors. Greatly improved solubility was observed for the expression of fusion proteins compared with the untagged protein expression. The trigger factor fused proteins showed the largest expression level among the chaperones. Bandscan analysis indicated that A113695 fused with trigger factor was a 95.5KD fusion protein, which was almost completely soluble, and the percentage of soluble fusion-protein in the total protein is 37.2%, higher than the percentage of untagged protein (7.1%). RB3348 fused with trigger factor was almost completely soluble too, and the percentage of soluble fusion-protein in the total protein is 53.0%, much higher than the percentage of untagged protein (4.4%).The positive-selection vector is one kind of vectors that directly selecte recombinant clones through antibiotics or chemical compounds. The biggest advantage is that the cloning efficiency that can be up to 100%. It was reported that bla missed the C-terminal W290 codon lost its ampicillin-resistance activity, while cloning a target gene carrying the respective sequence to complement this amino acid residue at the C-terminus of Bla into the vector would result in restoring the protein's function. In this report, we construced improved positive-selection protein expression vectors pPAE and pPAE-MBP by cloning Bla that lacks of several C-terminus amino acids into an expression vector that harboring the restriction site of TEV protease. Cloning experiments with E. coli aldolase gene (nanA) and human renin binding protein (hPnBP) showed that both pPAE and pPAE-MBP have 100% cloning efficiency, and the genes successfully expressed in E. coli.