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

Volpert M, Mangum JE, Jamsai D, D'Sylva R, O'Bryan MK, McIntyre P - Sci Rep (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.

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.

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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.
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f2: 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.

Mentions: Stable, inducible HEK 293 cell lines expressing human or mouse CRISP3 were generated. Following 96–120 hours of induction, human His-tagged CRISP3 constituted approximately 1.8% of total protein in the cell media (1.5 μg/ml by comparison with a CRISP3 standard). Protein stability and lack of toxicity was indicated by a proportional increase in CRISP3 concentration with induction time (Figure 2A). Deglycosylase treatment with Peptide -N-Glycosidase F (PNGase F) confirmed that like native CRISP3, the recombinant protein was secreted in unglycosylated and N-glycosylated forms (Figure 2B)19.


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

Volpert M, Mangum JE, Jamsai D, D'Sylva R, O'Bryan MK, McIntyre P - Sci Rep (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3936225&req=5

f2: 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.
Mentions: Stable, inducible HEK 293 cell lines expressing human or mouse CRISP3 were generated. Following 96–120 hours of induction, human His-tagged CRISP3 constituted approximately 1.8% of total protein in the cell media (1.5 μg/ml by comparison with a CRISP3 standard). Protein stability and lack of toxicity was indicated by a proportional increase in CRISP3 concentration with induction time (Figure 2A). Deglycosylase treatment with Peptide -N-Glycosidase F (PNGase F) confirmed that like native CRISP3, the recombinant protein was secreted in unglycosylated and N-glycosylated forms (Figure 2B)19.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Dept of Pharmacology, University of Melbourne, Parkville, VIC, Australia.

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.

Show MeSH