title: Solubility partner IF2 Domain I enables high yield synthesis of transducible transcription factors in Escherichia coli.
abstract: Since the discovery that somatic cells could be reprogrammed back to a pluripotent state through the viral expression of a certain set of transcription factors, there has been great interest in reprogramming using a safer and more clinically relevant protein-based approach. However, the search for an efficient reprogramming approach utilizing the transcription factors in protein form requires a significant amount of protein material. Milligram quantities of transcription factors are challenging to obtain due to low yields and poor solubility. In this work, we describe enhanced production of the pluripotency transcription factors Oct4, Sox2, Klf4, Nanog, and Lin28 after fusing them to a solubility partner, IF2 Domain I (IF2D1). We expressed and purified milligram quantities of the fusion proteins. Though the transcription factor passenger proteins became insoluble after removal of the IF2D1, the un-cleaved Oct4, Sox2, Klf4, and Nanog fusion proteins exhibited specific binding to their consensus DNA sequences. However, when we administered the un-cleaved IF2D1-Oct4-R9 and IF2D1-Sox2-R9 to fibroblasts and measured their ability to influence transcriptional activity, we found that they were not fully bioactive; IF2D1-Oct4-R9 and IF2D1-Sox2-R9 influenced only a subset of their downstream gene targets. Thus, while the IF2D1 solubility partner enabled soluble production of the fusion protein at high levels, it did not yield fully bioactive transcription factors.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Yang
authors.forename: John P
authors.initials: JP
authors.lastname: Welsh
authors.forename: Jieun
authors.initials: J
authors.lastname: Lee
authors.forename: John P
authors.initials: JP
authors.lastname: Cooke
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D000090062:Kruppel-Like Factor 4; D039881:Prokaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D039881:Prokaryotic Initiation Factor-2; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA