Our previous studies have shown that the holothurian Apostichopus japonicus produces a mannan-binding lectin (MBL-AJ) that possesses a unique specificity for carbohydrates, which allows its use for cancer antigen detection. In the present work, we report on the isolation of the gene encoding MBL-AJ and its heterologous expression in Escherichia coli cells. Expression of MBL-AJ was carried out under the control of an inducible promoter in the E. coli Top10/pQE-80L expression system. The recombinant MBL-AJ was purified by flow-through column metal-affinity chromatography. Optimal conditions for the refolding of recombinant MBL-AJ were selected. The “sandwich” ELISA method with an antibody against native MBL-AJ was used to determine the values of immunochemical cross reactivity of the native MBL-AJ and its recombinant forms that were obtained in different ways. The extent of immunochemical homology between native and recombinant MBL-AJ obtained under optimal conditions was 69%.
Russian Journal of Marine Biology – Springer Journals
Published: Mar 27, 2012
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