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Expression and purification of recombinant mouse CRISP4 using a baculovirus system
Avinash S Gaikwad1, Khai Lee Loh2, Anne E O'Connor1
1The School of Biological Sciences, Monash University, Clayton, Victoria, 3800, Australia.
Protein Expression and Purification
|November 24, 2019
Summary
Researchers developed a new method to produce soluble and correctly folded mouse Cysteine-rich secretory protein 4 (CRISP4). This breakthrough overcomes previous challenges, enabling further study of CRISP4
Area of Science:
- Reproductive Biology
- Protein Biochemistry
- Molecular Biology
Background:
- Cysteine-rich secretory protein 4 (CRISP4) is crucial for mammalian fertility and highly expressed in the male reproductive tract.
- CRISPs possess conserved structural domains, including the N-terminal CAP domain and C-terminal ion channel regulatory (ICR) domain.
- Previous recombinant protein expression attempts for CRISPs yielded misfolded proteins, aggregates, or low yields, hindering functional studies.
Purpose of the Study:
- To establish an efficient protocol for expressing and purifying recombinant mouse CRISP4.
- To overcome previous challenges in obtaining soluble and correctly folded CRISP proteins.
- To facilitate future functional investigations of CRISP4.
Main Methods:
- A three-step purification protocol was developed for mouse CRISP4 expression.
- High Five™ cells and a baculovirus expression system were utilized for protein production.
- Recombinant protein was validated using western blotting and structural characterization via Circular Dichroism (CD).
Main Results:
- The developed protocol successfully produced high yields of recombinant mouse CRISP4.
- The purified recombinant mouse CRISP4 was confirmed to be soluble and correctly folded.
- Western blotting and CD analysis validated the protein's identity and structural integrity.
Conclusions:
- A robust and efficient method for producing soluble, correctly folded recombinant mouse CRISP4 has been established.
- This protocol overcomes significant hurdles in CRISP protein purification.
- The availability of high-quality recombinant CRISP4 will enable detailed functional studies.
