Identification and Use of Zinc Finger Transcription Factors That Increase Production of Recombinant Proteins in Yeast and Mammalian Cells

Abstract

Randomized ZFP-TF libraries could induce a specific phenotype without detailed knowledge about the phenotype of interest because, theoretically, the libraries could modulate any gene in the target organism. We have developed a novel method for enhancing the efficiency of recombinant protein production in mammalian and microbial cells using combinatorial libraries of zinc finger protein transcription factors. To this end, we constructed tens of thousands of zinc finger proteins (ZFPs) with distinct DNA-binding specificities and fused these ZFPs to either a transcriptional activation or repression domain to make transcriptional activators or repressors, respectively. Expression vectors that encode these artificial transcription factors were delivered into Saccharomyces cerevisiae or HEK 293 cells along with reporter plasmids that code for human growth hormone (hGH) or SEAP (secreted alkaline phosphatase) (for yeast or HEK, respectively). Expression of the reporter genes was driven by either the cytomegalovirus (CMV) or SV40 virus promoters. After transfection, we screened the cells for increased synthesis of the reporter proteins. From these cells, we then isolated several ZFP-transcription factors (ZFP-TFs) that significantly increased hGH or SEAP synthesis and subjected these regulatory proteins to further characterization. Our results show that randomized ZFP-TF libraries are useful tools for improving the yield of heterologous recombinant protein both in yeast and mammalian cells.

Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells

“…Various methods have been developed to increase the yield and reduce the production costs of recombinant proteins, and in many studies human secreted alkaline phosphatase (SEAP) is utilized as a model product [15,16]. To see if MCRE can be utilized to enhance the production of recombinant proteins in stably transfected mammalian cells, we directly connected 3 or 7 copies of 5′-TTCCCCGCCG-3′ without spacer and placed them upstream of the CMV promoter in the pcDNA5/FRT vector expressing SEAP.…”

Transcriptional Activators of Human Genes with Programmable DNA-Specificity

“…These tailored TAL proteins (HuTALs) strongly induced reporter gene activation more than 30-fold and also endogenous human PUMA , IFNα1 , and IFNβ1 mRNA levels. Endogenous gene induction was considerably weaker (1.5-fold to 3.5-fold), a phenomenon which was also observed with artificial ZF transcription factors [35], [36]. The efficiency of TAL protein-dependent endogenous human gene induction in the two recent studies varied.…”

Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers

“…ATFs that induce drug resistance [171,172,174,221], cell differentiation [170] and antibody production [173] have been identified. Cell-surface markers [68], ICAM-1 (intercellular adhesion molecule-1) [163], VEGF (vascular endothelial growth factor) [60] and SEAP (secreted embryonic alkaline phosphatase) [222] have also served as gene targets. The Barbas group implemented an elegant selection strategy to retrieve functional ATFs that induce γ -globin expression.…”