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United States Department of Agriculture

Agricultural Research Service

Research Project: MICROBIAL CATALYSTS TO PRODUCE FUEL ETHANOL AND VALUE ADDED PRODUCTS Title: Functional Proteomic Workcell for High Volume Plasmid Preparations for Repeated in Vitro Protein Expression and High Throughput Screening to Identify Mutant Enyzmes for Use at Low Ph

Authors
item Hughes, Stephen
item Riedmuller, Steven - HUDSON CONTROL
item Mertens, Jeffrey
item Jordan, Douglas
item Li, Xin Liang
item Qureshi, Nasib
item Cotta, Michael
item Farrelly, Philip - HUDSON CONTROL
item Bischoff, Kenneth

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: September 18, 2005
Publication Date: September 18, 2005
Citation: Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Jordan, D.B., Li, X., Qureshi, N., Cotta, M.A., Farrelly, P.J., Bischoff, K.M. 2005. Functional proteomic workcell for high volume plasmid preparations for repeated in vitro protein expression and high throughput screening to identify mutant enyzmes for use at low pH [abstract]. Optimization High-throughput Cultures for Bioprocessing 2005. 13:3.

Technical Abstract: The possibility of examining the activity of enzymes at low pH has been explored using a plasmid-based functional proteomic robotic workcell. A novel mutagenesis paradigm was designed to alter the last four codons in CelF from the anaerobic fungus Orpinomyces PC-2, which code for the four amino acids at the amino end of the CelF enzyme. Plasmid DNA containing the mutagenized inserts can be produced in sufficient quantity for repeated in vitro transcription/translation in 96-well microplate-based expression reactions to measure activity of the mutant enzymes at low pH without need of in vivo expression systems. Screening for enhanced activity of the mutants is accomplished by spotting on an azo-carboxymethylcellulose plate.

Last Modified: 4/18/2014
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