Recombinant human sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) is expressed at high yield as an active homotetramer in baculovirus-infected insect cells

• Published in: Protein expression and purification Vol. 75; no. 1; pp. 104 - 113
• Main Authors: Lamson, David R., House, Alan J., Danshina, Polina V., Sexton, Jonathan Z., Sanyang, Khaddijatou, O’Brien, Deborah A., Yeh, Li-An, Williams, Kevin P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2011
• Subjects: Amino Acid Sequence; Animals; Baculoviridae - genetics; Baculovirus; Cell culture; Cell Line; Cloning, Molecular - methods; GAPDHS; Glyceraldehyde-3-Phosphate Dehydrogenases - chemistry; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics; Glyceraldehyde-3-Phosphate Dehydrogenases - isolation & purification; Glyceraldehyde-3-Phosphate Dehydrogenases - metabolism; Homotetramer; Humans; Insect cell; Male; Mass Spectrometry; Molecular Sequence Data; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; Protein Isoforms - metabolism; Protein Multimerization; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sperm; Spermatozoa - enzymology; Spodoptera - cytology; Spodoptera - genetics; Spodoptera - virology; Spodoptera frugiperda; GAPDHS; Homotetramer; Baculovirus; Insect cell; Sperm; Mass spectrometry
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2010.09.003

The sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS) isoform is a promising contraceptive target because it is specific to male germ cells, essential for sperm motility and male fertility, and well suited to pharmacological inhibition. However, GAPDHS is difficult to isolate from native sources and recombinant expression frequently results in high production of insoluble enzyme. We chose to use the Bac-to-Bac baculovirus-insect cell system to express a His-tagged form of human GAPDHS (Hu his-GAPDHS) lacking the proline-rich N-terminal sequence. This recombinant Hu his-GAPDHS was successfully produced in Spodoptera frugiperda 9 (Sf9) cells by infection with recombinant virus as a soluble, enzymatically active form in high yield, >35 mg/L culture. Biochemical characterization of the purified enzyme by mass spectrometry and size exclusion chromatography confirmed the presence of the tetrameric form. Further characterization by peptide ion matching mass spectrometry and Edman sequencing showed that unlike the mixed tetramer forms produced in bacterial expression systems, human his-GAPDHS expressed in baculovirus-infected insect cells is homotetrameric. The ability to express and purify active human GAPDHS as homotetramers in high amounts will greatly aid in drug discovery efforts targeting this enzyme for discovery of novel contraceptives and three compounds were identified as inhibitors of Hu his-GAPDHS from a pilot screen of 1120 FDA-approved compounds.