title: Cell-free production of transducible transcription factors for nuclear reprogramming.
abstract: Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target.
authors.affiliation: Department of Bioengineering, Stanford University, California 94305, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Yang
authors.forename: Kedar G
authors.initials: KG
authors.lastname: Patel
authors.forename: Jieun
authors.initials: J
authors.lastname: Lee
authors.forename: Yohannes T
authors.initials: YT
authors.lastname: Ghebremariam
authors.forename: H Edward
authors.initials: HE
authors.lastname: Wong
authors.forename: John P
authors.initials: JP
authors.lastname: Cooke
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051379:Mice; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA