Cloning, prokaryotic expression and characterization of lysine decarboxylase gene from Huperzia serrata.
- Author:
Ci DI
;
Jing LI
;
Yuntao TANG
;
Qingzhong PENG
- Publication Type:Journal Article
- MeSH:
Carboxy-Lyases;
biosynthesis;
genetics;
Cloning, Molecular;
Escherichia coli;
metabolism;
Genetic Vectors;
Huperzia;
enzymology;
genetics;
Lysine;
metabolism;
Plant Proteins;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2014;30(8):1299-1307
- CountryChina
- Language:Chinese
-
Abstract:
Huperzine A is a promising drug to treat Alzheimer's disease (AD). To date, its biosynthetic pathway is still unknown. Lysine decarboxylase (LDC) has been proposed to catalyze the first-step of the biosynthesis of huperzine A. To identify and characterize LDCs from Huperzia serrata, we isolated two LDC fragments (LDC1 and LDC2) from leaves of H. serrata by RT-PCR and then cloned them into pMD 19-T vector. Sequence analysis showed that LDC1 and LDC2 genes shared 95.3% identity and encoded the protein of 212 and 202 amino acid residues respectively. Thus, we ligated LDC genes into pET-32a(+) to obtain recombinant expressing vectors pET-32a(+)/LDC1 and pET-32a(+)/LDC2 respectively. We further introduced two expression vectors into Escherichia coli BL21(DE3) and cultured positive colonies of E. coli in liquid LB medium. After inducing for 4 hours with 260 μg/mL IPTG at 30 degrees C, soluble recombinant Trx-LDC1 and Trx-LDC2 were obtained and isolated for purification using a Ni-NTA affinity chromatography. We incubated purified recombinant proteins with L-lysine in the enzyme reaction buffer at 37 degrees C and then derived the reaction products using dansyl chloride. It was found that both Trx-LDC1 and Trx-LDC2 had decarboxylase activity, could convert L-lysine into cadaverine by way of thin layer chromatography assay. Further, bioinformatics analysis indicated that deduced LDC1 and LDC2 had different physicochemical properties, but similar secondary and three-dimensional structures.
