Cell-free production of transducible transcription factors for nuclear reprogramming

Department of Bioengineering, Stanford University, California 94305, USA.
Biotechnology and Bioengineering (Impact Factor: 4.13). 12/2009; 104(6):1047-58. DOI: 10.1002/bit.22517
Source: PubMed


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.

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Available from: Jieun Lee, Oct 06, 2015
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    • "One study employing CPP-fused Nanog protein further reports that recombinant Nanog could translocate to the nucleus (Yang et al., 2009); however, no further data was shown to demonstrate the functionality of the protein. "
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    ABSTRACT: A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation.
    Stem Cell Research 03/2014; 12(3):680-689. DOI:10.1016/j.scr.2014.02.006 · 3.69 Impact Factor
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    • "Cell-free protein synthesis (CFPS) with bacterial extracts has been successfully used in this laboratory to produce a large variety of active proteins. These proteins include, but are not limited to, metalloproteins such as ferredoxin (26) and [FeFe] hydrogenase (27), virus-like particles (28), membrane proteins (29,30), transcription factors (31) and proteins containing nnAAs (32–35). Recently, the cell-free accumulation of 700 mg/l of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was demonstrated at 100-litre scale (36). "
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    ABSTRACT: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.
    Nucleic Acids Research 04/2013; 41(11). DOI:10.1093/nar/gkt226 · 9.11 Impact Factor
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    • "are fusion peptides, each with a reprogramming factor , a linker, and a cell transduction domain (Yang et al., 2009). These CPPs exhibit cognate DNA-binding activity, rapidly translocate across the plasma and nuclear membranes, uniformly transduce nearly all cells in the treated wells, and exert transcriptional control on known downstream target genes. "
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    ABSTRACT: Retroviral overexpression of reprogramming factors (Oct4, Sox2, Klf4, c-Myc) generates induced pluripo-tent stem cells (iPSCs). However, the integration of foreign DNA could induce genomic dysregulation. Cell-permeant proteins (CPPs) could overcome this limitation. To date, this approach has proved ex-ceedingly inefficient. We discovered a striking differ-ence in the pattern of gene expression induced by viral versus CPP-based delivery of the reprogram-ming factors, suggesting that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In gain-and loss-of-function studies, we find that the toll-like receptor 3 (TLR3) pathway enables effi-cient induction of pluripotency by viral or mmRNA approaches. Stimulation of TLR3 causes rapid and global changes in the expression of epigenetic modi-fiers to enhance chromatin remodeling and nuclear reprogramming. Activation of inflammatory path-ways are required for efficient nuclear reprogram-ming in the induction of pluripotency.
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