Obtaining and refolding a recombinant mannan-binding lectin from the holothurian Apostichopus japonicus

Obtaining and refolding a recombinant mannan-binding lectin from the holothurian Apostichopus... 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%. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Russian Journal of Marine Biology Springer Journals

Obtaining and refolding a recombinant mannan-binding lectin from the holothurian Apostichopus japonicus

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Publisher
SP MAIK Nauka/Interperiodica
Copyright
Copyright © 2012 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Freshwater & Marine Ecology
ISSN
1063-0740
eISSN
1608-3377
D.O.I.
10.1134/S1063074012010130
Publisher site
See Article on Publisher Site

Abstract

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%.

Journal

Russian Journal of Marine BiologySpringer Journals

Published: Mar 27, 2012

References

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