Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

Abstract

While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

Figure 1. CRISP domain organisation and tertiary structure.

(A) CRISP domain organisation and (B) homology models of mouse and human CRISP3 based on the crystal structure of Natrin. The CRISP domain is shown in blue/aqua; disulfide bonds by white/yellow linkages. N-glycosylation is represented by high mannose (mouse CRISP3) and complex (human CRISP3) glycans (ball and stick models).

Figure 2. HEK 293 cells efficiently secrete unglycosylated and N-glycosylated recombinant human CRISP3.

(A) Western blot with human CRISP3 antibody showing CRISP3 induction. (B) PNGase F treatment selectively affects the larger CRISP3 variant in both native (human seminal plasma, first two lanes) and recombinant (‘rCRISP3-His', last two lanes) protein. Full length gels are available under supplementary data.

Figure 3. CRISP3 is enriched 15-fold following CM cellulose exchange.

(A) Silver-staining at various purification stages. Lane 1: starting material (250 μl conditioned media), lane 2: CM cellulose flow through, lanes 3–10: CM cellulose exchange gradient elutions (CRISP3 elutes in lanes 7–8, representing 110 and 240 mM NaCl respectively). (B) CRISP3 gel filtration to over 60% purity. (C) Mouse CRISP3 purified by the same method (two bands, 26 and 33 kDa). The 48 kDa contaminant is alpha-enolase. (D) Gel filtration absorbance trace with human CRISP3 peak indicated by the arrow. All gels were run under the same conditions.

Figure 4. Recombinant human CRISP3 binds human and bovine A1BG.

(A) Gel analysis of proteins purified on recombinant CRISP3-coated cyanogen bromide resin showed CRISP3 interaction with A1BG from both human (lane 3, ~85 kDa band) and bovine (lane 3, ~65 kDa band) serum, as confirmed by mass spectrometry. Elution control samples were loaded as follows; lane 1: uncoated beads incubated with serum, lane 2: CRISP3-coated beads incubated with PBS, lane 3: CRISP3-coated beads incubated with serum. In the case of human serum, all eluted proteins in lane 3 other than A1BG bound non-specifically to the beads (compare with lane 1). (B) Denatured CRISP3 (lane 3) was unable to bind A1BG. Full length gels are available under supplementary data; gels were run under the same conditions.

Figure 5. Recombinant mouse CRISP3 is glycosylated at N99 and N113.

Western blotting with His tag antibody showed glycosylated human and mouse CRISP3-His to be 29 and 33 kDa respectively (A). (B) Peptide pre-absorption of mouse CRISP3 antibody. (C) Mouse CRISP3 N99Q and N113Q mutant proteins were smaller than wild type (WT) mouse CRISP3 (PNGase F untreated samples); N156Q was unaffected. Two mutants unable to undergo glycosylation (N99Q + N113Q and N99Q + N113Q + N156Q) were of relatively low abundance. ‘NT' represents non-transfected HEK cell media. Full length gels are available under supplementary data; gels were run under the same conditions.

Figure 6. Mouse CRISP3 is incompletely digested by Endo H.

(A) Mouse CRISP3 wild type, N99Q and N113Q are partially digested by Endo H (E). Complete digestion with PNGase F (P) is included for size comparison. (B) A size comparison of mouse and human CRISP3 (mCRISP3 and hCRISP3, respectively) glycans (square brackets). Full length gels are available under supplementary data; gels were run under the same conditions.

Figure 7. CRISP3 from whole mouse salivary gland is unglycosylated.

Western blot comparison of wild type salivary gland CRISP3 with recombinant mouse CRISP3 glycosylated at two sites (wild type, lane 1), one site (N99Q, lane 2) and unglycosylated (N99Q + N113Q + N156Q, lane 3). Full length gel is available under supplementary data.