title: Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.
abstract: Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5.
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
authors.forename: Amanda
authors.initials: A
authors.lastname: Blythe
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
authors.forename: Sanjika
authors.initials: S
authors.lastname: Gunasekara
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
authors.forename: James
authors.initials: J
authors.lastname: Walshe
authors.affiliation: School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Joel P
authors.initials: JP
authors.lastname: Mackay
authors.affiliation: Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA.
authors.forename: Grant A
authors.initials: GA
authors.lastname: Hartzog
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Electronic address: alice.vrielink@uwa.edu.au.
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D061805:Transcription Elongation, Genetic; D035602:Transcriptional Elongation Factors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C080991:SPT4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035602:Transcriptional Elongation Factors; D025801:Ubiquitin; C069940:SPT5 transcriptional elongation factor
keywords: Expression and purification; KOW domain; Spt4/5; Ubiquitin tag; pHUE