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Competitive, immunometric assay for fusion protein quantification: Protein production prioritization

Cho, Hyun-Hee, Alderman, Edward, Kreder, Natasha, Caro, Roxana Garcia, Leong, Kristen, Miller, Michael F., Hill, W. Adam G., Pandey, Pramod
Analytical biochemistry 2014 v.446 pp. 1-8
acoustics, antibodies, drugs, genetic vectors, immunoblotting, labor, microparticles, polyacrylamide gel electrophoresis, prioritization, protein synthesis, purification methods, quantitative analysis, recombinant fusion proteins, screening, staining
Effective drug discovery demands the availability of microgram to gram quantities of high-quality protein encoded by novel transcripts. Protein expression vectors designed for large-scale protein production often include one or more specific tags to such transcripts, to simplify the purification of the targeted protein. Optimization of the complex expression and purification process requires the evaluation of multiple expression candidate clones to identify a production-suitable construct in terms of quality and final protein yield. Efficiency of the entire expression screening process is typically assessed by direct visualization of the banding patterns from whole-cell lysates on SDS–PAGE gels, by direct staining and/or immunoblotting, using antibodies against the tag or the protein of interest. These techniques, generally run under denaturing conditions, have proven to be only marginally predictive of the purification yield and authentic folding for native proteins. Small-scale, multiparallel affinity purification followed by SDS–PAGE analysis is more predictive for expression screening; however, this approach is labor intensive and time consuming. Here we describe the development of an alternative expression efficiency assessment technique, designed to evaluate the accessibility of affinity tags expressed with the desired fusion proteins, using acoustic membrane microparticle assay technology on the ViBE protein analysis workstation.