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.
N1 - Funding Information:
This work was supported by the Crohn’s and Colitis Foundation of America, the NIH (R01 DK070855 to L.V.H.), and a Burroughs Wellcome Career Award in the Biomedical Sciences to L.V.H. We thank Karen Lewis and Dr. Philip Thomas for help with circular dichroism studies and members of the Hooper laboratory for constructive criticism of the manuscript.
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
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U2 - 10.1016/j.pep.2006.01.014
DO - 10.1016/j.pep.2006.01.014
M3 - Article
C2 - 16504538
AN - SCOPUS:33646871553
SN - 1046-5928
VL - 48
SP - 151
EP - 159
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -