Expression and purification of recombinant mouse CRISP4 using a baculovirus system

Avinash S. Gaikwad, Khai Lee Loh, Anne E. O'Connor, Hugh H. Reid, Moira K. O'Bryan

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

Original languageEnglish
Article number105543
Number of pages6
JournalProtein Expression and Purification
Volume167
DOIs
Publication statusPublished - 1 Mar 2020

Keywords

  • CAP proteins
  • CRISP4
  • Cysteine-rich secretory protein
  • Epididymal CRISP
  • Insect cell expression

Cite this

@article{07df92797ff5430ab155a82b62f27d77,
title = "Expression and purification of recombinant mouse CRISP4 using a baculovirus system",
abstract = "Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.",
keywords = "CAP proteins, CRISP4, Cysteine-rich secretory protein, Epididymal CRISP, Insect cell expression",
author = "Gaikwad, {Avinash S.} and {Lee Loh}, Khai and O'Connor, {Anne E.} and Reid, {Hugh H.} and O'Bryan, {Moira K.}",
year = "2020",
month = "3",
day = "1",
doi = "10.1016/j.pep.2019.105543",
language = "English",
volume = "167",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Elsevier",

}

Expression and purification of recombinant mouse CRISP4 using a baculovirus system. / Gaikwad, Avinash S.; Lee Loh, Khai; O'Connor, Anne E.; Reid, Hugh H.; O'Bryan, Moira K.

In: Protein Expression and Purification, Vol. 167, 105543, 01.03.2020.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Expression and purification of recombinant mouse CRISP4 using a baculovirus system

AU - Gaikwad, Avinash S.

AU - Lee Loh, Khai

AU - O'Connor, Anne E.

AU - Reid, Hugh H.

AU - O'Bryan, Moira K.

PY - 2020/3/1

Y1 - 2020/3/1

N2 - Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

AB - Cysteine-rich secretory protein 4 (CRISP4) is a member of the CAP superfamily protein, is highly expressed in the male reproductive tract and is required for optimal mammalian fertility. CRISPs are characterized by the presence of 16 conserved cysteine residues which forms 8 disulphide bond spread across the N-terminal CAP domain, a hinge region and a C-terminal ion channel regulatory (ICR) domain. Previous attempts to purify recombinant CRISPs as a group have resulted in misfolded and/or insoluble recombinant proteins, protein aggregates or unusable low protein yield. Thus, defining the functions of CRISPs have been impeded. In this study, we report a three-step purification protocol for expression and purification of mouse CRISP4 protein in High Five™ cells using a baculovirus expression system. Recombinant mouse CRISP4 was recognized by western blotting and structurally characterized using Circular Dichroism (CD). Using the protocol described herein, we generated high yields of soluble and correctly folded recombinant mouse CRISP4.

KW - CAP proteins

KW - CRISP4

KW - Cysteine-rich secretory protein

KW - Epididymal CRISP

KW - Insect cell expression

UR - http://www.scopus.com/inward/record.url?scp=85075316362&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2019.105543

DO - 10.1016/j.pep.2019.105543

M3 - Article

VL - 167

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

M1 - 105543

ER -