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 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.

Figure 1

Schematic diagram of full-length human GAPDHS and the recombinant human his-GAPDHS expressed in insect cells. The sperm isozyme possesses a proline-rich domain at the N-terminus that is not present in the somatic GAPDH isozyme. In human GAPDHS, this extension comprises a 19 amino acid conserved sequence, 3 amino acid intervening proline cysteine region and a 42 amino acid proline-rich region (50% proline) from residues 27–67 [3]. Numbering in this figure refers to the sequence for human sperm-specific gyceraldehyde-3-phosphate dehydrogenase (GAPDHS) (GenBank Accession ID: NP_055179.1). For the form of GAPDHS expressed in insect cells herein, the N-terminal extension containing the proline-rich region was deleted (residues 1–68) and replaced by the 30 amino acid FastBac vector sequence which included a hexahistidine purification tag and TEV protease cleavage sequence.

Figure 2

Sequence alignment between recombinant human his-GAPDHS and predicted Spodoptera frugiperda GAPDH. For simplification, residue numbering begins at 1 for the recombinant his-GAPDHS. The 30 residue FastBac tag is shown italicized. The sequence for Spodoptera frugiperda (Sf) GAPDH was from SF9L02522-Contig2, a contig assembled from the SPODOBASE EST database (see supplemental Fig. 1 and supplemental Table 1). Regions of identity are shown with a light grey background and regions of similarity with a dark grey background.

Figure 3

Optimization of recombinant human his-GAPDHS expression in Sf9 cells. A. MOI analysis of his-GAPDHS expression. Sf9 cells were infected with recombinant GAPDHS virus at the indicated MOI and samples of media (M) and cell lysates (L) taken 96 h post-infection were analyzed by SDS-PAGE. Protein bands resulting from infection with the recombinant virus carrying the gene for GAPDHS are indicated by arrows. B. Western blot analysis of cell lysates at the indicated MOI from experiment A above using a penta-his HRP conjugated antibody. C. Small scale expression in 25 cm² T-flask of Hu his-GAPDHS in Sf9 cells and Ni-NTA affinity purification. Lane M, Novex prestained protein markers; lane 1, cell lysate from recombinant virus infected S9 cells, lane 2, mock infected Sf9 cells, lane 3, Ni-NTA resin unbound fraction, lane 4, Ni-NTA resin wash fraction, lane 5, Ni-NTA resin eluted fraction showing a major band at ~40 kDa corresponding to human his-GAPDHS.

Figure 4

SDS-PAGE/Western blot analysis of human his-GAPDHS purified by Ni²⁺-NTA affinity chromatography. A. Coomassie-Blue stained gel, lane M, Novex prestained protein markers; lane 1, Hu his-GAPDHS, lane 2, Silver-stained gel of Hu his-GAPDHS, lane 3, Western blot of Hu his-GAPDHS with polyclonal antibody raised to residues 159–194 of mouse GAPDHS [8], lane 4, Western blot of Hu his-GAPDHS with penta-his HRP conjugated antibody. B. Coomassie-Blue stained gel; lane M, Novex prestained protein markers and indicated amounts of purified Hu his-GAPDHS.

Figure 5

MALDI-TOF MS spectra of purified recombinant human GAPDHS. MALDI-TOF analysis was performed using α-cyano-4-hydroxycinnamic acid as the matrix. The peaks at 40,740.22, 20,350.85 and 10,180.70 are single-, double-, and quadruple-charge species.

Figure 6

Size exclusion chromatography of purified human his-GAPDHS. A. Chromatogram of purified Hu his-GAPDHS loaded on a Superose 12 column at 0.5 ml/min in 50 mM potassium phosphate pH 7.0, 150 mM NaCl buffer. B. A calibration curve was created using the elution volumes (V_e) of the following standard proteins; ferritin (M_r 440,000), aldolase (M_r 158,000), conalbumin (M_r 75,000), ovalbumin (M_r 44,000), carbonic anhydrase (M_r 29,000), ribonuclease A (M_r 13,700). K_av values were calculated [19] using a void volume (V_o) of 7.4 ml and included volume of 19.9 ml (V_i). The molecular size of recombinant human his-GAPDHS was estimated to be ~130,000 (arrow).

Figure 7

Pilot high throughput screen using Hu his-GAPDHS enzyme. The Prestwick compound collection was pre-spotted (0.5 μl) in 384-well plates and the Hu his-GAPDHS enzyme assay carried out in a final volume of 50 μl as described in materials and methods. A. Scatterplot showing percent inhibition for compounds screened and with hits shown as diamonds above 30% inhibition. B. Histogram of screening data showing a normal distribution and, C. Box plot representing quantiles and outliers.