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

Protein Expression and Purification
Heather L CashLora V Hooper

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 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 int...Continue Reading

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Citations

Apr 17, 2010·Inflammation Research : Official Journal of the European Histamine Research Society ... [et Al.]·Domenico ViterboMartin H Bluth
Dec 17, 2008·Proceedings of the National Academy of Sciences of the United States of America·Shipra VaishnavaLora V Hooper
Dec 20, 2008·The Journal of Biological Chemistry·Sohini MukherjeeLora V Hooper
Aug 26, 2006·Science·Heather L CashLora V Hooper
Aug 2, 2012·American Journal of Physiology. Endocrinology and Metabolism·Maria PetropavlovskaiaLawrence Rosenberg
Feb 9, 2008·Annals of Surgical Oncology·Kaori HayashiJun-ichi Ogawa
Jun 23, 2011·PloS One·Christian StelterDirk Bumann
Apr 13, 2010·Proceedings of the National Academy of Sciences of the United States of America·Rebecca E LehotzkyLora V Hooper
Jan 22, 2015·Immunity·Sohini Mukherjee, Lora V Hooper
Aug 25, 2009·Protein Expression and Purification·Guoyong HuWei Han
Jan 27, 2009·Biochemical and Biophysical Research Communications·Ryota AbeKouhei Tsumoto
Aug 30, 2008·Biophysical Chemistry·Makoto NakakidoKouhei Tsumoto
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Jun 11, 2017·Scientific Reports·Laia BonjochDaniel Closa
Jan 12, 2021·International Reviews of Immunology·Yuwen CaoZhaoliang Su

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