The Agouti Signaling Protein ASIP Darren A Thompson

The Agouti Signaling Protein (ASIP) Darren A. Thompson and Glenn L. Millhauser University of California at Santa Cruz Grow BL 21 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 M 6 H MK 25 -131 YY ASIP M 6 H MK 25 -87 Absorbance 214 nm Absorbance 280 nm non-agouti Mutate two residues to tyrosine for improved oxidation HPLC reduced ASIP after Nickel Column lethal yellow agouti Once gene in vector transform into e. coli BL 21 and have bacteria make protein oxidized reduced 5 disulfides 0 disulfides Air oxidation of ASIP appears as banded gray Absorbance 214 nm Absorbance 280 nm HPLC oxidized ASIP for bioactivity tests PCR evidence gene is in vector. No gene PCR fragment 262 bp T 7 primers/ 0 bp ASIP primers 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.