Expression, Purification, and Assembly of the Functional Human Stearoyl-CoA Desaturase Complex using Cell-Based and Cell-Free Technologies Michael A. Goren.

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 UniversityYaeta 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 long-chain 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 His8-MBP variants were tested

• Also applied to soluble domain of flavin-containing cytb5 reductase

In situ delivery of functional fl-cytb5

Liposome floating methodSobrado, 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, Spatz, Corcoran, Rogers, Setlow, Redline. PNAS, 71, 4565-4569 (1974).

Strittmatter’s heroic effort

• 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 re-suspended to equal volume

• Estimated yield 0.2 - 2.6 mg/mL by Caliper electrophoresis

• Co-fractionation with Hsp70?

What about wheat germ lysate?

germ lysate.

Element Lot 1 Lot 2 Fatty acid Concentration (µM)

Mg (mM) 4.5 2.3 16:0 500P (mM) 54.1 44.7 18:0 1.8S (mM) 52.6 53.3 cis ∆9 18:1 15K (mM) 170 156 cis ∆11 18:1 1.2

Ca (mM) 0.2 0.2 18:2(n-6) 37Fe (µM) 26.3 23.3Ni (µM) 2.4 2.2Co (µM) 0.2 0.1Cu (µM) 45.3 40.8Zn (µM) 265 234Se (µM) 1.8 1.6

Table 1. IPC-AES analysis of wheat germ Table 2. Fatty acid contentof wheat germ lysate.

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  Supplement Brij-35 Triton X-100 Triton X-100 CHAPS Liposome

hSCD1 2.26 2.23 – 2.14 – 2.35

sigma 1 receptor 1.26 1.39 1.26 – 1.01 1.52

bacteriorhodopsin 0.8 – – – 1.17

GFP 1.05 0.91 – 0.92 0.69 0.94

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, annotatedMan, Miyazaki, Chu and Ntambi. JBC. 281, 1251-1260 (2006).

A recent SCD topology model, annotatedMan, 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