Cloning, Expression, And The Anti-tumor Activity Of Recombinant MAP30 In Escherichia Coli

MAP30 (Momordica anti-HIV protein of 30kD) is a 30kD single-stranded, type-I ribosome inactivating protein purified from Momordica charantia(bitter melon). It is capable of acting against multiple stages of the viral life cycle on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase, cell-free ribosome-inactivation and antimicrobial activities. And what's more, MAP30's cytotoxicity is specific to tumor-transformed or viral-infected cells. It shows no adverse effects on normal cells, making it a candidate for clinical applications.We have cloned the gene encoding the mature MAP30 protein from the total DNA of bitter melon by PCR. After sequenced, the MAP30 gene was cloned into the vector pET-30a to construct the expression vector pET-30a-MAP30 with the N-terminal 6His-tag. The pET-30a-MAP30 vector was transformed into competent E. coli BL21 (DE3) by calcium chloride transformation method, and the recombinant was identified by PCR analysis. The recombinant MAP30 was expressed mainly in soluble form with lmmol/L IPTG induction in E. coli, and its amount reached highest at 30°C up to 56% of total bacterial protein.. According to the N-terminal fusion 6his-tag, the fusion protein then was purified by affinity chromatography. The last purified protein was about 37.2% of the total supernatant protein, and the zymotic productivity was 250 mg/L. Western blot analysis indicated that the recombinant MAP30 had the antigenicity to Rabbit anti-His-tag polyclonal antibody. The anti-tumor activity and cytotoxicity assay of recombinant MAP30 showed that it exhibits dose-dependent inhibition to 3T3 and S-180 lines, and the dose required for 50% inhibition (ID50) were 50ug/ml and 3.0 mg/ml, respectively. Little cytotoxic effect on WI-38 was found under the same assay condition.Our results demonstrate that the recombinant MAP30 expressed in E. coli is active to tumor cells, it provides an abundant source of homogeneous material for the study of its other biological property and further development of genetic engineering products.