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J Biotechnol. 2013 Mar 10;164(1):112-20. doi: 10.1016/j.jbiotec.2012.11.015. Epub 2012 Dec 5.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

Author information

1
DSM Biotechnology Center, Alexander Fleminglaan 1, 2613AX Delft, The Netherlands. Michiel.Akeroyd@DSM.com

Abstract

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery. Here, we present a sensitive, generic workflow for high throughput screening of successful microbial protein over-expression in microtiter plates containing a complex matrix based on mass spectrometry techniques. MALDI-LTQ-Orbitrap screening followed by principal component analysis and peptide mass fingerprinting was developed to obtain a throughput of ∼12,000 samples per week. Alternatively, a UHPLC-MS(2) approach including MS(2) protein identification was developed for microorganisms with a complex protein secretome with a throughput of ∼2000 samples per week. TCA-induced protein precipitation enhanced by addition of bovine serum albumin is used for protein purification prior to MS detection. We show that this generic workflow can effectively reduce large expression libraries from fungi and bacteria to their minimal size by detection of successful protein over-expression using MS.

PMID:
23220267
DOI:
10.1016/j.jbiotec.2012.11.015
[Indexed for MEDLINE]

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