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

Abstract

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 Nterminal 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, >35mg/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.

Development and Implementation of a High Throughput Screen for the Human Sperm-Specific Isoform of Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDHS)

“…Expression of full-length GAPDHS in E. coli has proven difficult in quantities sufficient for screening a large number of compounds [ 7 ]. Substantially better yields and activities were obtained by elimination of the proline-rich N-terminal region [ 1 ] that is not required for enzyme activity [ 22 ]. When typical E. coli strains were used for expression, mass spectrometry analyses confirmed that mouse GAPDHS and the host bacterial GAPDH formed mixed tetramers, resulting in the co-purification of bacterial and recombinant enzymes (data not shown).…”

“…Typical yields were ~13 mg of purified GST-GAPDHS per 6L E. coli culture. Hu GST-GAPDHS protein purified from E. coli was further characterized by analytical size exclusion chromatography as previously described [ 22 ]. GST-GAPDHS was applied to a Superose 12 column and eluted as a mixture of three forms (Fig.…”

“…Protein concentrations were estimated by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Analytical size exclusion chromatography was carried out on a Superose 12 column (GE Healthcare) as previously described [ 22 ].…”

“…Some compounds were also tested by dose response in the presence of detergent (0.01% Tween-20 final concentration). In some cases, compounds were also tested by dose response against a form of human histag-GAPDHS expressed in the baculovirus/insect cell system [ 22 ]. Buffer conditions were as described above and his-GAPDHS was used at a final concentration of 60 nM in the assay.…”

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm

“…This proline-rich part confers a change in biochemical properties of the enzyme. While GAPDH is an abundant cytoplasmic protein, highly soluble and easy to purify and crystallize, the sperm GAPDHS protein becomes highly insoluble, slowly migrating in the gel, and numerous attempts to determine the crystal structure of the whole protein failed due to its properties [ 9 - 11 ]. Its crystal structure without the N-terminal part was found and shows high similarity to the somatic enzyme.…”