The Agouti Signaling Protein (ASIP)
Darren A. Thompson and Glenn L. Millhauser
University of California at Santa Cruz
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.
807.8x16-16 = 12908.8
861.4x15-15 = 12906.0
922.8x14-14 = 12905.2
993.7x13-13 = 12905.1
1076.4x12-12 = 12904.8
1174.2x11-11 = 12905.2
1291.4x10-10 = 12904.0
1434.9x9-9 = 12905.1
861.4
807.6
922.4
ASIP M6H
MK25-131 YY
993.7
1076.4
ASIP M6H
MK25-87
1174.2
1291.4
1434.9
Absorbance 214 nm
Absorbance 280 nm
500
non-agouti
1000
1500
2000
m/z
Mutate two residues to tyrosine for improved oxidation
6
8
10
12
14
16
time (min)
HPLC reduced ASIP after Nickel Column
A214 t=0
A214 t=2 hr
A214 t=overnight
lethal
yellow
agouti
oxidized
reduced
5 disulfides
0 disulfides
18
19
20
21
22
23
24
25
26
time (min)
Air oxidation of ASIP
Once gene in vector transform into e. coli BL21 and have
bacteria make protein
12895
922.1
860.7
806.9
992.9
11000
11500
12000
12500
13000
13500
14000
reconstructed mass
1075.5
1173.1
1290.5
1433.6
500
1000
1500
2000
m/z
appears as
banded gray
0
5
10
15
Absorbance 214 nm
Absorbance 280 nm
20
25
30
35
time (min)
HPLC oxidized ASIP for bioactivity tests
PCR evidence gene is in vector. No gene PCR
fragment 262 bp T7 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.