| Summary: | 碩士 === 國立中正大學 === 化學工程所 === 94 === A recombinant strain E. coli BL21 harboring pGEX-1λT Neu5Ac aldolase-5R was constructed by the technology of gene cloning to over-express a double-tagged fusion protein, GST-N-acetyl-D-neuraminic acid aldolase-(Arg)5 (GST-Neu5Ac aldolase-5R). The gene construction has been confirmed able to express target protein by the analysis of gel electrophoresis and DNA sequencing. In this study, the induction cultivation and cell-lysis process for the recovery of over-expressed proteins from two recombinant strains, E. coli BL21 harboring pGEX-1λT Neu5Ac aldolase-5R and E. coli BL21 harboring pGEX-2TKGlcNAc 2-epimerase-5D, were investigated in order to find the best conditions for the achievement of highest protein productivity. Both the expression level of soluble proteins and their activities in the crude extracts were of great concern. The influence of non-ionic detergent Triton X-100 concentration on the cell lysis was also studied. Results indicated that the highest amount of soluble proteins was obtained when 1% of Triton X-100 was added to harvested cells for lysis.
On the study of induction conditions, different concentrations of IPTG (0.01, 0.1 and 1.0 mM) were added to the cell culture for the induction of protein over-expression. The highest level of fusion proteins was observed when 0.01 mM IPTG was used to induce the expression of target proteins. Enzymatic activities of the doubled-tagged fusion proteins GST-GlcNAc 2-epimerase-5D and GST-Neu5Ac aldolase-5R were determined to be 0.043 and 0.034 U/mg, respectively.
The fusion protein tagged with gluthione S-transferase (GST) was able to be purified through the affinity between GST tag and GSH. By this way, the recovery of GST-GlcNAc 2-epimerase-5D and GST-Neu5Ac aldolase-5R based on enzymatic activity were 25 and 41.1 %, respectively.
Using contiguous residues (5R) at the C-terminus, fusion proteins could be sequentially purified and immobilized. Also, the specific activity of GST-Neu5Ac aldolase-5R could be advanced from 4.15 to 7.74 U/mg through the immobilization procedure. To produce sialic acid (Neu5Ac), a coupling reaction of double-tagged GlcNAc 2-epimerase and Neu5Ac aldolase was performed using 100 mM GlcNAc and 100 mM pyruvate as substrates in a 100-ml mixture containing 4 mM ATP and 8 mM MgCl2 (pH 7.0). When GST-GlcNAc 2-epimerase-5D and GST-Neu5Ac aldolase-5R, both in the soluble form, were present in the reaction mixture for incubation at 30℃ for 24 h, 0.21 mmoles of Neu5Ac produced. As a comparison, when the reaction mixture was incubated with immobilized GST-GlcNAc 2-epimerase-5D and GST-Neu5Ac aldolase-5R at 30℃ for 24 h, we could harvest 0.27 mmoles of Neu5Ac at the final. These results suggested that double-tagged fusion proteins could retain their enzymatic activities in the immobilized form.
|
|---|
