Poster Abstract

February 5, 2010

A Robust Technique for ASIP Production and Refolding

Michael Mendoza
MARC U*STAR Fellow Summer Research Site: UC Santa Cruz

The information gathered from studying the genetics of mammalian coat color origin has provided insight into the details of many biological processes. The gains from this field of study have furthered our understanding of signal transduction, body weight regulation, linkage analysis and DNA transcription. The Agouti Signaling Protein (ASIP) is a protein found in skin cells and regulates a switch between melanin pigment colors. It is an antagonist to the Melanocortin 1 Receptor (MC1R) which responds to alpha-melanocyte stimulating hormone (a-MSH). ASIP also antagonizes a homologous hypothalamic receptor MC4R in some strains of inbred mice. These lethal yellow mice with pronounced obesity, hyperphagia, and linear growth are explained by the genetic fusion between ASIP and the ubiquitously expressed RNA binding protein. A search of the Expressed Sequence Database discovered that a similar protein, the Agouti Related Protein (AgRP), antagonizes MC4R but not MC1R. As the focus shifted over the years towards studying AgRP, many questions about the nature of ASIP have been left unanswered In order to study the biological processes ASIP is involved in, the bio-molecule must first be produced in measurable quantities and in its native state. There are many techniques available to do this, such as baculovirus infection of insect cells and solid phase protein synthesis. At the University of California Santa Cruz, a new robust technique for the purification of ASIP using bacterial expression in BL-21 E.coli cells and column chromatography has been developed. This method of protein purification has been coupled with an air oxidation procedure to ensure proper refolding and disulfide pair formation of its ten oxidizable cysteine residues. Michael Mendoza University of California Santa Cruz Dr. Millhauser Graduate Student – Darren Thompson