HPLC reduced ASIP after Nickel Column

1. The Agouti Signaling Protein (ASIP) Darren A. Thompson and Glenn L. Millhauser University of California at Santa Cruz Absorbance 214 nm Absorbance 280 nm appears as banded gray Grow BL21 in LB Miller broth Induce with IPTG Lyse cells Affinity purify Ni-NTA Agouti signaling protein (ASIP) was identified more than 15 years ago as a secreted protein that binds to melanocortin receptors throughout the body and affects hair color and body weight. To better understand its pharmacologic and physiologic properties, I have developed an approach to generate large amounts of biologically active ASIP using a biosynthetic and chemical refolding approach. ASIP M6H MK25-131 YY ASIP M6H MK25-87 non-agouti Mutate two residues to tyrosine for improved oxidation HPLC reduced ASIP after Nickel Column lethal yellow agouti reduced 0 disulfides oxidized 5 disulfides Air oxidation of ASIP Once gene in vector transform into e. coli BL21 and have bacteria make protein Absorbance 214 nm Absorbance 280 nm HPLC oxidized ASIP for bioactivity tests Initial tests of biologic activity (affinity for the Melanocortin 1 receptor) suggest that the refolded protein has approximately 20 - 50 fold less activity than anticipated. Current experiments are directed at understanding whether this is due to alterations in tertiary structure or post-translational modification. PCR evidence gene is in vector. No gene PCR fragment 262 bp T7 primers/ 0 bp ASIP primers