Cho, Hyunhee, Alderman, Ed, Kreder, Natasha, Garcia Caro, Roxana, Leong, Kristen, Hill, William and Pandey, Pramod (2014) Immunometric AMMP assay for rapid analysis of expression constructs for protein production. Competitive, immunometric assay for fusion protein quantification: Protein production prioritization.

Abstract

Drug discovery efforts are highly dependent on production of high-quality drug target proteins. For this purpose the protein expression laboratories are tasked with the production of microgram-to-gram quantities of proteins encoded by novel transcripts. To accelerate this process, most expression laboratories now rely on the generation of chimeric fusion proteins, in which the novel transcript is encoded with one or more specific tags, in order to simplify the purification of the targeted protein (Hunt, 2005; Walls & Loughran, 2011; Young et al., 2012). Optimization of conditions for expression and purification is a complex process. It often requires analysis of large numbers of expression clones to identify a suitable construct for final protein expression which would yield ample amount of protein. Efficiency of the entire expression screening process is typically assessed by direct visualization of the banding patterns on SDS-PAGE gels by staining and/or immunoblots, using antibodies against the tag or the protein of interest in the whole cell lysates. These techniques, generally run under denaturing conditions, have proved to be only marginally predictive of the purification yield for the native protein. Small scale multi-parallel affinity purification followed by running SDS-PAGE gels is more predictive for expression screening (Nguyen et al., 2004), however this approach is very labor intensive and time consuming. Here we describe the development and utility of an alternate efficiency assessment technique, designed to evaluate the accessibility of affinity tags expressed with the desired fusion proteins, using Acoustic Membrane MicroParticle (AMMP®) assay technology on the ViBE® Protein Analysis Workstation.

Item Type:	Article
Date Deposited:	02 May 2016 23:45
Last Modified:	02 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/9822