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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: High-Throughput Screening of Cellulase F Mutants from Multiplexed Plasmid Sets Using An Automated Plate Assay on a Functional Proteomic Robotic Workcell

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

Submitted to: Proteome Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 19, 2006
Publication Date: May 2, 2006
Citation: Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Li, X., Bischoff, K.M., Qureshi, N., Cotta, M.A., Farrelly, P.J. 2006. High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell. Proteome Science. 4:10.

Interpretive Summary: A functional proteomic assay on a plasmid-based robotic workcell is used to identify mutants of cellulase F that have improved activity at low pH. The assay was used to screen the recombinant CelF mutants expressed from cultures that were multiplexed eight to a well in a 96-well plate. Individual clones were isolated from the multiplexed cultures using the robotic workcell for picking and inoculating single cultures from the glycerol stocks linked to the multiplexed wells, preparing plasmid, producing recombinant protein, and assaying for enzyme activity. Four CelF mutants with improved activity were identified. The multiplexing method using an integrated automated workcell for high-throughput screening with a functional proteomic assay increases the number of clones that can be screened and allows rapid identification of optimized clones.

Technical Abstract: The field of plasmid-based functional proteomics requires the rapid evaluation of proteins expressed from plasmid libraries. Automation is essential since large sets of clones are involved. To date no integrated robotic platform is available to carry out the entire process from creation of plasmid libraries, to expressing cloned genes, and finally to functional testing of expressed proteins. We describe here a functional proteomic assay using a plasmid-based robotic workcell to identify mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2, that have improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. The robotic picker from the workcell was used to inoculate medium in a 96-well deep-well plate, combining eight transformants per well into a multiplexed set, and the plate incubated on the workcell. Using the liquid handler component of the workcell, plasmid was prepared from the multiplexed culture and used for in vitro transcription/translation. The expressed recombinant proteins were screened for improved activity and stability in an azo carboxymethylcellulose plate assay. Five multiplexed cultures were identified as containing mutants having improved activity. Individual clones were then isolated from the multiplexed cultures using the workcell for inoculating single cultures from stock spread plates, preparing plasmid, producing recombinant protein, and assaying for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of four improved CelF mutants, one possessing both increased thermal stability and higher activity at pH 4 than wild-type CelF. The multiplex method using an integrated automated workcell for high-throughput screening in a functional proteomic assay increases the numbers of clones that can be screened and allows for rapid identification of optimized clones.

Last Modified: 7/24/2014
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