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

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• 作者：Kyung-Soon Park ; Wongi Seol ; Hyo-Young Yang ; Seong-Il Lee ; Sung Keun Kim ; Ryuk Jun Kwon ; Eui-Joong Kim ; Young-Hoon Roh ; Baik Lin Seong ; and Jin-Soo Kim
• 刊名：Biotechnology Progress
• 出版年：2005
• 出版时间：June 2005
• 年：2005
• 卷：21
• 期：3
• 页码：664 - 670
• 全文大小：173K
• 年卷期：v.21,no.3(June 2005)
• ISSN：1520-6033

文摘

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