High-Throughput Protein Production (HTPP): A Review of Enabling Technologies to Expedite Protein Production

Abstract

Recombinant protein production plays a crucial role in the drug discovery process, contributing to several key stages of the pathway. These include exploratory research, target validation, high-throughput screening (HTS), selectivity screens, and structural biology studies. Therefore the quick and rapid production of high-quality recombinant proteins is a critical component of the successful development of therapeutic small molecule inhibitors. This chapter will therefore attempt to provide an overview of some of the current "best-in-class" cloning, expression, and purification strategies currently available that enhance protein production capabilities and enable greater throughput. As such the chapter should also enable a reader with limited understanding of the high-throughput protein production (HTPP) process with the necessary information to set up and equip a laboratory for multiparallel protein production.

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

“…High-throughput protein expression and purification maintain a pivotal role in structural biology (reviewed in [6]). The polyHis-tag IMAC purification process has enabled numerous structural studies; in fact, more than 60% of the all protein structures that exist include a polyHis-tag [78].…”

“…While contemporary reports have included several overviews of available affinity tags for protein detection or purification [1][2][3][4], solubility enhancement [5], tag removal [1], or applications [6,7] -in this comprehensive review, we provide an extensive summary of various tags and established purification strategies. We describe several design aspects for these tags that should be considered for recombinant fusion protein expression, including amino acid composition and size; N-or C-terminal fusions, used individually or in tandem; as well as optimized tags, techniques, and buffer conditions that lead to purified protein.…”

A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

“…The rising of protein-protein interaction studies as well as the constantly increasing demand of proteins in drug discovery programs have brought the need for high-throughput protein expression and automation to the foreground [1][2][3][4][5]. The need for recombinant proteins with post-translational modifications has fostered the development of mammalian cell-based expression systems rather than prokaryotes and lower eukaryotes [6].…”

“…Over the last 10 years, major improvements of the technique leading to better yields have made transient gene expression a competitive alternative to the establishment of stable cell lines as it shortens the time required to obtain the target protein in sufficient amount from several months to a few weeks only [7][8][9][10][11][12]. The capacity to quickly express recombinant proteins in mammalian cells by transfection is thus of great benefit not only for large-scale applications but also for highthroughput expression for functional screening and crystallization purposes [3,[11][12][13][14][15].…”

Mammalian cell culture density determination using a laser through-beam sensor

“…Automated workflows for both mammalian transient protein production and protein expression from stable cell lines are used in many laboratories [1,2]. We have previously described fully automated cell culture workflows performed on GNF System's (CA, USA) protein expression and purification platform (PEPP) for the production and purification of endogenousand recombinant proteins from mammalian cells such as hybridomas, FreeStyle 293-F, (ThermoFisher Scientific #R79007, MA, USA) and CHO [3].…”