Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli.
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 RegIIIgamma 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 beta-strands which is typical of C-type lectins. Finally, both RegIIIgamma 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.
Cash
Heather L
HL
Center for Immunology, The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Blvd., Dallas, TX 75390, USA.
Whitham
Cecilia V
CV
Hooper
Lora V
LV
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001704:Biopolymers / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008358:Mannose / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D000073718:Pancreatitis-Associated Proteins; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D001704:Biopolymers; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; D011506:Proteins; C000612141:REG3A protein, human; D011994:Recombinant Proteins; C088549:RegIII protein, mouse; D008358:Mannose