Fusion Expression Vector And Applications As Well As Double-coupled Catalytic Synthesis Of N- Acetyl Enzyme Neuraminidase -D-

Many heterologous proteins exhibit as insoluble, biologically inactive inclusion body form when overexpressed in Escherichia coli, thus hampering the functional study. Fusion expression is a practical means to achieve soluble expression. To obtain general fusion vectors, five fusion tags, including mutated maltose binding protein (mMBP), small ubiquitin-related modifier (SUMO), initiation factor2(IF2), N utilization substance A (NusA) and glutathione S-transferase (GST); three chaperones, including GroEL, danK, TF were each cloned into E. coli expression vector pET30a(+) to obtain eight dual-tagged fusion expression vectors. These expression vectors contain the same cloning sites and stuffer fragment, with tobacco etch virus protease (TEV) digestion site engineered after carboxyl terminus of fusion tag. Four N-acetyl-D-glucosamine2-epimerase genes were cloned into mMBP fusion vector, after IPTG induction, SDS-PAGE analysis showed that high-level expression and nearly all soluble proteins were obtained for all four fusion proteins, in contrast, nearly no soluble form were observed when the genes were expressed without Tag. Fusion protein could be isolated with Ni-NTA chromatography, and tag may be removed after TEV digestion. For mMBP and GST fusion proteins, affinity resin and Ni-NTA chromatography can be combined for target protein isolation. Expected digestion patterns were achieved after TEV digestion of four fusion proteins. The newly developed expression vectors have potential for the broad applications in protein expression, isolation and function study.N-acetyl-D-neuraminic acid(Neu5Ac) and its derivates are a very important group of biomolecules, because these sugars occupy the terminal position in numerous macromolecules and are involved in many biological and pathological phenomena. Moreover, Neu5Ac is the key intermediate in the synthesis of the anti-flu drug zanamivir. So Neu5Ac is expensive and in short demand worldwide. Enzymatic catalysis of Neu5Ac production is very popular due to the advantages of easy, low-cost, and environmentally friendly operation under mild condition. The procedure consists of two steps:first, N-acyl-D-glucosamine2-epimerase(AGE) catalyzes the transformation of N-acetyl-D-glucosamine (GlcNAc) into N-aceyl-D-mannosamine (ManNAc); second, the condensation of ManNAc and pyruvate into Neu5Ac through N-acetyl-D-neuraminic acid Aldolase (Ald). We tried to increase the solubility of several AGEs by fusion expression, which may also enhance the AGE activity. In addition, through homology search with the amino acid sequence of pig renin binding protein(pRnBP) as a query, BT0453gene was amplified from Bacteroides thetaiotaomicron VPI-5482and cloned, expressed and purified in Escherichia coli. The highest activity of AGE was determined by measuring Neu5Ac concentration achieved from N-acetyl-D-glucosamine (GlcNAc) and pyruvate using GlcNAc2-epimerase and Neu5Ac aldolase (nanA) as the biocatalysts. The results showed that BT0453protein has the highest activity, the reason is probably that the expressed soluble protein takes about80%of the total protein. In a100ml whole cell biocatalysis system,112.8g1-1of Neu5Ac was achieved corresponding to a molar conversion rate of45.6%after a12h reaction. A higher conversion rate of55.8%was obtained within12h reaction when pyruvate was supplemented, which is nearly20%higher than the existing literature data and builds the foundation for the industrial production of Neu5Ac.