Expression and purification of the recombinant GAPDHS, sperm-specific protein of energy metabolism

0380452 - BTÚ 2013 CZ eng A - Abstract
Dorosh, Andriy - Děd, Lukáš - Margaryan, Hasmik - Pěknicová, Jana
Expression and purification of the recombinant GAPDHS, sperm-specific protein of energy metabolism.
XVII. symposium českých reprodukčních imunologů s mezinárodní účastí. Praha: Biotechnologický ústav v. v. i. AVČR, 2011 - (Pěknicová, J.). s. 64-64
[XVII. symposium českých reprodukčních imunologů s mezinárodní účastí. 26.05.2011-29.05.2011, Žďár nad Sázavou]
R&D Projects: GA ČR(CZ) GA523/09/1793; GA ČR(CZ) GA523/08/H064; GA MŠMT(CZ) 1M06011; GA MZd(CZ) NS10009
Institutional research plan: CEZ:AV0Z50520701
Keywords : recombinant protein * GAPDHS
Subject RIV: CE - Biochemistry

Testis-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is a key enzyme of glycolysis in sperm cells. It is the 408 aa long protein, which forms catalytically active tetramers and differs from somatic enzyme with N-terminal proline-rich part. Male mice that lack GAPDHS gene are infertile due to sperm motility disorders. In this work, full-length recombinant protein fused with either 6xHis or GST tag was expressed in E.coli BL21 strain using Gateway® technology and subsequently affinity purified. GST-fused GAPDHS (75kDa) had both higher expression levels and solubility in comparison with 6xHis tagged (47kDa) protein. Then, we found that MoAb 6E4, recognizing postacrosomal part of the sperm head in immunofluorescence staining, detects the recombinant GAPDHS after Western blotting. It is in agreement with the data that immunoprecipitated protein with 6E4 antibody from sperm cell lysate was identified as GAPDHS. Previously, GAPDHS protein was localized exclusively to principal piece of the sperm tail. In future, function of this protein in postacrosomal region of sperm head will be studied.
Permanent Link: http://hdl.handle.net/11104/0211162