[Cloning and expression of N-acetyl-D-neuraminic acid aldolase in Escherichia coli].

OBJECTIVE

To obtain the Escherichia coli strains expressing N-Acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase).

METHODS

The gene (nanA) coding Neu5Ac aldolase was cloned from Escherichia coli C600, and the recombinant plasmid was sequenced and expressed in Escherichia coli.

RESULTS

Sequencing data revealed that the open reading frame was 894 bp and predicted to encode a protein consisting of 298 amino acids. The patterns of SDS-PAGE showed that the purified enzyme protein as a single protein band with a molecular weight of 33 kD, which was consistent with those reported in the reference. In the recombinant plasmid pRY1, the expression of nanA gene was controlled by the lac promoter with the induction of IPTG or lactose. The plasmid pRY3 was constructed, in which the nanA gene ws controlled by the tac promoter. The protein of Neu5Ac aldolase was constitutively expressed using the recombinant strain, E.coli DH5 alpha/pRY3 without induction of IPTG or lactose. The crystal was finally obtained with the efficiency of 90.2% of Neu5Ac. The HPLC indicated that the Neu5Ac crystal prepared in this experiment was same as Simga product.

CONCLUSION

The protein products expressed by two recombinant strains E.coli BL21(DE3)/pRY1 and DH5 alpha/pRY3 has the characteristics of Neu5Ac.