Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli

Nature Biotechnology
E L SchneiderF Baneyx

Abstract

Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm. While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation. Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice. Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein. Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart. Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

References

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Citations

Oct 6, 1999·Current Opinion in Biotechnology·F Baneyx
Mar 12, 2010·Journal of the American Chemical Society·Weibin ZhouFrançois Baneyx
Nov 6, 2004·Nature Biotechnology·François Baneyx, Mirna Mujacic
Jan 31, 2012·Applied and Environmental Microbiology·Joaquin Seras-FranzosoElena García-Fruitós
Jan 29, 2009·Microbial Cell Factories·Olga KolajJ Gerard Wall
Aug 27, 2005·Journal of Cellular Physiology·Catherine F DelostrinosJames A Bassuk
Mar 7, 2008·Critical Reviews in Biotechnology·S Venketesh, C Dayananda

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