[Cloning and heterologous expression of laccase genes vv-lac1 and vv-lac6 from Volvaria volvacea].

OBJECTIVE

To clone the full-length cDNAs of two laccase genes, vv-lac1 and vv-1ac6, from Volvaria volvacea, verify their encoded proteins with laccase activity and develop a heterologous expression and protein purification system for V. volvacea laccase genes.

METHODS

The full-length cDNAs were cloned with rapid amplification of cDNA ends (RACE) technology and carried out in silico analysis. After modified by removing the sequence encoding signal peptide and adding the sequence encoding His-tag at 3' ends, the cDNAs were cloned into pPIC9K vector. The resulting constructs were transformed into Pichia pastoris GS115 for heterologous expression. The recombinant proteins were purified with Ni columns and the laccase activity were detected with ABTS assay.

RESULTS

The full-length cDNAs of vv-lac1 and vv-lac6 are 1,599 bp and 1,554 bp, and contain19 and 15 exons, respectively. The predicted molecular weights of the proteins encoded by vv-lac1 and vv-lac6 are 57.3 kDa and 56.3 kDa, respectively. The predicted isoelectric points are 4.73 and 5.62, respectively. Both proteins are extracellular. The recombinant proteins RBvvlacland RBvvlac6 are 70 kDa, which may be modified by posttranslational modification. The solutions of the two recombinant proteins eluted by 150 mmol/L imidazole eluent have the highest laccase activity levels (333.17 U/L and 227.63 U/L).

CONCLUSION

The proteins encoded by the laccase genes vv-lac1 and vv-lac6 from V. volvacea have laccase activity, the heterologous expression and protein purification system developed in this study is suitable for future studies of other laccase genes from V. volvacea or other fungi.