Production and characterization of recombinant transducible transcription factors [electronic resource]
Keywords: Bioengineering.

The overall objective of this thesis was to produce and characterize recombinant transducible (able to enter cells) transcription factors (TFs). TFs are complex, difficult-to-make proteins that regulate gene expression and cell fate. Thus, we wanted to deliver transducible TFs exogenously to cells to change gene expression and alter cell fate. Ectopic expression of a set of pluripotency-regulating TFs (Oct4, Sox2, Klf4, cMyc, Lin28, and Nanog) using viral vectors can reprogram adult cells to pluripotency. The resultant cells are pluripotent and can self-renew indefinitely, just like embryonic stem cells. These induced pluripotent stem cells (iPSCs) hold enormous potential for patient-specific cell-based therapies and disease models. However, viruses integrate foreign DNA into the host cell genome, causing insertional mutagenesis and genomic instability which renders the cell unsuitable for clinical use. A non-viral method of iPSC generation is necessary in order to bring iPSCs from the bench to bedside. Direct delivery of the TFs as recombinant proteins offers one avenue for non-viral iPSC generation. Conjugation of an arginine-rich protein transduction domain (PTD) to the TF cargo enables intracellular delivery of the exogenously administered fusion protein. The overall goal of this multi-investigator project was to develop a protein-based method for iPSC generation using purified recombinant TFs fused with a nona-arginine (R9) PTD. The first step is to efficiently produce TF proteins that are active in controlling downstream target gene expression. Thus, my thesis focuses on the upstream portion of the overall project, the production and characterization of the recombinant transducible TFs. TFs are difficult to produce in live cell cultures due to aggregation and product toxicity. Thus, we used E. coli cell-free protein synthesis (CFPS) to produce these transducible TFs. CFPS decouples protein synthesis from maintenance of host cell health to permit production of toxic proteins and slows the translation rate to favor proper protein folding. Using CFPS, we made R9-Oct4, R9-Sox2, R9-Klf4, R9-cMyc, R9-Lin28, and R9-Nanog. Taking advantage of the flexibility offered by CFPS, we addressed protein truncation and solubility problems to produce full-length and soluble TFs. We then showed that R9-Oct4, R9-Sox2, and R9-Nanog exhibit specific binding to their respective consensus DNA sequence probes. We also demonstrated that R9-Nanog enters cells and that R9-Sox2 both enters the cell and upregulates the expression of its downstream gene targets. Though we made full-length and soluble TFs, the soluble yields were still low. The open nature of CFPS allows us to perturb protein production conditions by adding excipients to enhance soluble production. However, it is often not clear if the excipients facilitate the production of fully functional proteins. Therefore, we developed a filter microplate assay for quantitative analysis of DNA binding (one function of the TFs) to potentially be used as a screen for identifying protein production conditions that enable production of soluble and active protein. We validated this assay by showing specific DNA binding, affinity, and capacity for CFPS-produced Sox2 and Nanog. We also attempted to improve soluble transducible TF production in live E. coli cultures using a solubility fusion partner, the first domain of the E. coli initiation factor 2 (IF2D1). IF2D1 improved both total and soluble expression of the IF2D1-TF-R9 fusion proteins, but when the IF2D1 was removed, the TF-R9 cargo became insoluble even though the un-cleaved IF2D1-TF-R9 exhibited the expected DNA binding activity. Unfortunately, un-cleaved IF2D1-Oct4-R9 and IF2D1-Sox2-R9 were not fully bioactive when administered to cells. They only influenced the expression of a subset of their downstream target genes, suggesting that while the IF2D1 solubility partner improved soluble TF expression, it did not yield functional TFs. In parallel, we sought to quantify the intracellular delivery of the transducible TFs. Using radioactively labeled R9-Sox2 and Sox2, we showed that both proteins bind equally well to the outside of target fibroblast cells, but only R9-Sox2 enters the cell. This observation was confirmed using immunohistochemistry to visualize protein delivery as well as quantitative PCR to show that R9-Sox2 influences intracellular gene expression while Sox2 does not. The prevailing hypothesis for PTD-mediated cellular entry is fluid phase endocytosis of the cationic PTD fusion proteins bound on the cell surface. Our results provide evidence to support a competing hypothesis in the literature that binding of the PTD fusion to the cell surface is not sufficient for internalization. Originally, we sought to quantify both the intracellular delivery and localization of 14C leucine-labeled R9-Sox2. However, we reached the detection limit of 14C leucine radiation at the whole cell level. Thus, an alternative quantification method was necessary to measure endosomal entrapment, cytoplasmic delivery, and nuclear localization. We developed a sandwich enzyme-linked immunosorbent assay (ELISA) for this purpose. We hypothesized that picomolar sensitivity was required to detect the low levels of R9-Sox2 delivered to the nucleus. We identified an antibody pair that was able to detect picomolar concentrations of purified R9-Sox2. But, this sandwich ELISA exhibited a high degree of non-specific binding when it was performed in a mock experimental context by spiking known amounts of purified R9-Sox2 into cytoplasmic and nuclear lysates. We troubleshot non-specific binding by exploring various antibodies and assay conditions, but were not able to reduce non-specific binding to the level that enabled picomolar detection. However, we did identify a condition that enabled nanomolar detection of R9-Sox2 in undiluted cytoplasmic and nuclear lysates, a nearly 40-fold improvement over the sensitivity of 14C leucine radiation. In summary, this work described the production and characterization of recombinant transducible TFs to support a protein-based approach for iPSC generation. Using CPFS, we developed a platform for producing recombinant TFs as well as tools and methods for characterizing TF activity and intracellular localization. These studies also displayed the flexibility and potential offered by CFPS for engineering and producing complex fusion proteins.