Expression, Purification, and Assembly of the
Functional Human Stearoyl-CoA Desaturase
Complex using Cell-Based and Cell-Free
Technologies
Michael A. Goren and Brian G. Fox
Department of Biochemistry, College of Agricultural and Life Sciences, University of
Wisconsin, Madison WI 53706
NIGMS U54 GM074901
JL Markley, PI, GN Phillips and BG Fox, Co-Investigators
NIH GM50853, BG Fox
NSF East Asia and Pacific Summer Institutes Fellowship, MA Goren
Acknowledgements
CESG Staff:
David Aceti, Arash Bahrami, Lenka Bittova, Lai Bergeman, E. Sethe Bergie, Christopher
Bianchetti, Craig Bingman, Eduard Bitto, Claudia Cornilescu, Ronnie Frederick, Brian Fox,
Michael Goren, Katarzyna Gromek, Leigh Grundhoefer, Teagan Hayes, Andrew Larkin,
Elena Levin, Betsy Lytle, Shin-Ichi Makino, John Markley, Yuko Matsubara, Karl Nichols,
Xiaokang Pan, Francis Peterson, George N. Phillips, Jr. Mike Popelars, John Primm, Sarata
Sahu, Kory Seder, Donna Troestler, Frank Vojtik, Brian Volkman, Gary Wesenberg, Russell
Wrobel, and Zsolt Zolnai
University of Wisconsin Undergraduates:
Erin Bulleit, Michael Cassidy, Breanne Cyr, Colin Diner, Theresa Filarsky, Margaret Harteau,
Soyoon Hwang, Sydney Junion, Michael Kern, Heather Kindrachuk, Benjamin Jyhhan Kuo,
Haibo Li, Christopher Lindholm, Yun Liu, Steven Marmer, Kathryn Nix, Young Ju Oh, Lane
Oling, Joseph Taft, Jonathan Wagner, Nicholas Wild, Kevin Ziolo, Andrew Moriarity, and
Samantha Schmechel
Dane County Youth Apprentice Program:
Stuart Ballard, Zachery Kunkel, Joshua Perry, and Sharon West
Cell-Free Science and Technology Research Center, Ehime University
Yaeta Endo and Yuzuru Tozawa
On the stability of proteins…
“There are no unstable proteins, only unstable
biochemists…”
Irwin C. Gunsalus
Mammalian desaturases
• Add double bonds to longchain fatty acids
• Help to control membrane
composition
• Numerous diseases
associated with primary lipid
metabolism
Stearoyl-CoA desaturase complex
• A highly regulated enzyme
required for biosynthesis of
mono-unsaturated fatty acids
• cyt b5 reductase, full-length
cyt b5, SCD
• Implicated in obesity,
diabetes, heart disease, skin
disorders, etc
pVP56 expression of fl-cytb5
• Expression of the soluble,
non-functional domain as
His8-tagged protein
• Full-length His8 and His8MBP variants were tested
• Also applied to soluble
domain of flavin-containing
cytb5 reductase
In situ delivery of functional fl-cytb5
Liposome floating method
Sobrado, Goren, James, Amundsen, Fox. Protein Express Purif 58, 229-241, (2008).
Stearoyl-CoA desaturases
• Two isoforms in humans
– Tissue-specific expression
• Polytopic membrane
proteins
• 8-His motif
• Diiron active site
Strittmatter’s heroic effort
Strittmatter, Spatz, Corcoran, Rogers, Setlow, Redline. PNAS, 71, 4565-4569 (1974).
• Fifty well-fed, 30-day old Sprague-Dawley rats were starved,
then fed, then starved…
• Livers were sliced, diced, spun, soaped, salted, filtered,
mashed, washed, and…
• After 14 days in the cold room, ~5 mg of pure mSCD1 was
obtained
Regulated expression of SCD in vivo
• Control points in yeast
–
–
–
–
Transcriptional
Translational
Post-translational
Proteosome degradation
• Best expression by Western
blotting, is ~100 ng/mL
An alternative, cell-free translation
•
White dots show creatine
kinase, 700 µg/mL in the soluble
fraction
•
Stars show membrane
desaturases from pellets resuspended to equal volume
•
Estimated yield 0.2 - 2.6 mg/mL
by Caliper electrophoresis
•
Co-fractionation with Hsp70?
What about wheat germ lysate?
Table 1. IPC-AES analysis of wheat germ
germ lysate.
Element
Lot 1
Lot 2
Mg (mM)
P (mM)
S (mM)
K (mM)
Ca (mM)
Fe (µM)
Ni (µM)
Co (µM)
Cu (µM)
Zn (µM)
Se (µM)
4.5
54.1
52.6
170
0.2
26.3
2.4
0.2
45.3
265
1.8
2.3
44.7
53.3
156
0.2
23.3
2.2
0.1
40.8
234
1.6
Table 2. Fatty acid content
of wheat germ lysate.
Fatty acid
16:0
18:0
cis Ж9 18:1
cis Ж11 18:1
18:2(n-6)
Concentration (µM)
500
1.8
15
1.2
37
Cell-free translation with additives
Table 3. Protein Expression as Determined by Capillary Electrophoresis (mg/mL)
No
0.1% w/v 0.5% w/v
0.7% w/v 0.5% w/v
К
Supplement Brij-35 Triton X-100 Triton X-100 CHAPS Liposome
hSCD1
2.26
2.23
Р
2.14
Р
2.35
sigma 1 receptor
1.26
1.39
1.26
Р
1.01
1.52
bacteriorhodopsin
0.8
Р
Р
Р
1.17
GFP
1.05
Р
0.92
0.69
0.94
0.91
Translation of fl-cytb5 with liposomes
Translation of hSCD1 with liposomes
Combining hSCD1 and cytb5-fl
Addition of purified cytb5-fl to lysate
Co-translation
Enzymatic activity
A recent SCD topology model, annotated
Man, Miyazaki, Chu and Ntambi. JBC. 281, 1251-1260 (2006).
A recent SCD topology model, annotated
Man, Miyazaki, Chu and Ntambi. JBC. 281, 1251-1260 (2006).
Sobrado, Kaul, and Fox, in preparation.
Summary
• Wheat-germ cell free translation gives high level
expression of a difficult class of proteins
• Expressed proteins are transferred to liposomes with
high efficiency, possibly with some biological
assistance
• There are many simple options available for the
assembly of multi-protein complexes
• Catalytic function has been demonstrated for human
SCD1, an important biomedical complex