Cloning, expression and purification of fructose-2,6-bisphosphatase gene CpF2KP in papaya

Abstract:

Papaya, which is mainly cultivated in the southeastern region of China, is one of the four famous fruits in Lingnan. It is favored by people because of its edible and medicinal value. Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (F2KP) is a unique bifunctional enzyme with a kinase domain and an esterase domain that catalyzes the synthesis and degradation of fructose-2,6-bisphosphate (Fru-2,6-P₂), an important regulator of glucose metabolism in organisms. In order to study the function of the gene CpF2KP encoding the enzyme in papaya, it is particularly important to obtain the target protein. In this study, the coding sequence (CDS) of CpF2KP, with a full-length of 2 274 bp, was got from the papaya genome. The amplified sequence of full-length CDS was cloned into the vector PGEX-4T-1 which was double digested with EcoR I and BamH I. The amplified sequence was constructed into a prokaryotic expression vector by genetic recombination. After exploring the induction conditions, the results of SDS-PAGE showed that the size of the recombinant GST-CpF2KP protein was about 110 kDa. The optimum IPTG concentration and temperature for CpF2KP induction were 0.5 mmol/L and 28℃, respectively. The purified sin[A28] gle target protein was obtained after purifying the induced CpF2KP protein. In addition, the expression level of this gene was detected in different tissues, and showed that the gene was expressed at the highest level in seeds and the lowest in pulp. This study provides an important basis for further revealing the function of CpF2KP protein and studying the involved biological processes of this gene in papaya.