Abstract
BackgroundTwo sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione S-transferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R).ResultsBoth fusion proteins were overexpressed in Escherichia coli and retained enzymatic activity. The fusions were designed so their surfaces were charged under enzyme reaction conditions, which allowed isolation and immobilization in a single step, through a simple capture with either an anionic or a cationic exchanger (Sepharose Q or Sepharose SP) that electrostatically bound the 5D or 5R tag. The introduction of double tags only marginally altered the affinity of the enzymes for their substrates, and the double-tagged proteins were enzymatically active in both soluble and immobilized forms. Combined use of the fusion proteins led to the production of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).ConclusionDouble-tagged gene fusions were overexpressed to yield two enzymes that perform sequential steps in sialic acid synthesis. The proteins were easily immobilized via ionic tags onto ionic exchange resins and could thus be purified by direct capture from crude protein extracts. The immobilized, double-tagged proteins were effective for one-pot enzymatic production of sialic acid.
Highlights
Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions
This paper describes the construction of gene fusions for expressing GlcNAc 2-epimerase and Neu5Ac aldolase, both with an N-terminal glutathione S-transferase (GST) tag, and a C-terminal tag of five contiguous aspartate residues (5D) on GlcNAc 2-epimerase and five contiguous arginine residues (5R) on Neu5Ac aldolase
Under the inducing conditions indicated in Methods, E. coli BL21 harboring the gene fusion produced a significant level of doubletagged GlcNAc 2-epimerase when grown to an OD600 of approximately 1.0 and treated with IPTG to induce gene expression
Summary
Two sequential enzymes in the production of sialic acids, N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N-acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), were overexpressed as double-tagged gene fusions. Both were tagged with glutathione Stransferase (GST) at the N-terminus, but at the C-terminus, one was tagged with five contiguous aspartate residues (5D), and the other with five contiguous arginine residues (5R). N-acetyl-D-neuraminic acid (Neu5Ac) is the most abundant terminal carbohydrate in glycoconjugates (glycoproteins and glycolipids) and has great commercial value. It is an acidic sugar with a prominent role in numerous biological activities, including anti-viral and anti-bacterial defensive functions. GlcNAc 2-epimerase has been found in mammals (humans, rats and pigs) and in the unicellular cyanobacterium Synechocystis sp
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