Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli

Heather L. Cash, Cecilia V. Whitham, Lora V. Hooper

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIγ and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of β-strands which is typical of C-type lectins. Finally, both RegIIIγ and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.

Original languageEnglish (US)
Pages (from-to)151-159
Number of pages9
JournalProtein Expression and Purification
Volume48
Issue number1
DOIs
StatePublished - Jul 2006

Fingerprint

Escherichia coli
Purification
Proteins
C-Type Lectins
Hot isostatic pressing
Carbohydrates
Ligands
Mannans
Size exclusion chromatography
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Mannose
Circular Dichroism
Chromatography
Inflammatory Bowel Diseases
Recombinant Proteins
Gel Chromatography
Mass spectrometry
Arginine
Gastrointestinal Tract
Cations

Keywords

  • C-type lectins
  • Carbohydrate binding
  • Inflammatory bowel disease
  • Mucosal injury
  • Regenerating protein family

ASJC Scopus subject areas

  • Biochemistry

Cite this

Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli. / Cash, Heather L.; Whitham, Cecilia V.; Hooper, Lora V.

In: Protein Expression and Purification, Vol. 48, No. 1, 07.2006, p. 151-159.

Research output: Contribution to journalArticle

@article{322896ec56a641d8a608a68365671039,
title = "Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli",
abstract = "The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIγ and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of β-strands which is typical of C-type lectins. Finally, both RegIIIγ and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.",
keywords = "C-type lectins, Carbohydrate binding, Inflammatory bowel disease, Mucosal injury, Regenerating protein family",
author = "Cash, {Heather L.} and Whitham, {Cecilia V.} and Hooper, {Lora V.}",
year = "2006",
month = "7",
doi = "10.1016/j.pep.2006.01.014",
language = "English (US)",
volume = "48",
pages = "151--159",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli

AU - Cash, Heather L.

AU - Whitham, Cecilia V.

AU - Hooper, Lora V.

PY - 2006/7

Y1 - 2006/7

N2 - The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIγ and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of β-strands which is typical of C-type lectins. Finally, both RegIIIγ and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.

AB - The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIγ and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of β-strands which is typical of C-type lectins. Finally, both RegIIIγ and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.

KW - C-type lectins

KW - Carbohydrate binding

KW - Inflammatory bowel disease

KW - Mucosal injury

KW - Regenerating protein family

UR - http://www.scopus.com/inward/record.url?scp=33646871553&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646871553&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2006.01.014

DO - 10.1016/j.pep.2006.01.014

M3 - Article

VL - 48

SP - 151

EP - 159

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -