The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production

Abstract

The production of high yields of soluble recombinant protein is one of the main objectives of protein biotechnology. Several factors, such as expression system, vector, host, media composition and induction conditions can influence recombinant protein yield. Identifying the most important factors for optimum protein expression may involve significant investment of time and considerable cost. To address this problem, statistical models such as Design of Experiments (DoE) have been used to optimise recombinant protein production. This review examines the application of DoE in the production of recombinant proteins in prokaryotic expression systems with specific emphasis on media composition and culture conditions. The review examines the most commonly used DoE screening and optimisation designs. It provides examples of DoE applied to optimisation of media and culture conditions.

The Statistical Optimisation of Recombinant β-glucosidase Production through a Two-Stage, Multi-Model, Design of Experiments Approach

“…Recombinant β-glucosidases from different sources, heterologously expressed in prokaryotic and eukaryotic systems have typically utilised the traditional One Factor At a Time (OFAT) method to optimise yield [18]. However, this classical approach requires a significant number of experiments and fails to account for interactions between variables, which can result in low yields [8,31]. In this study, the production of S. griseus recombinant β-glucosidase has been optimised, instead, by Design of Experiments with the emphasis on optimising parameters that affected expression.…”

“…In an attempt to address this issue of poor production yields, this study applied a statistical approach, Design of Experiments (DoE), to enhance the production of a recombinant β-glucosidase. The application of DoE to optimise protein production has been recently reviewed [8,9]. Enhanced production of recombinant β-glucosidase, following DoE, has been detailed for recombinant β-glucosidases from a variety of sources, such as Pichia pastoris [3], A. niger HN-2 [10] and A. niger [11].…”

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

“…We have applied RSM, which allows evaluation of several factors in parallel with a minimum number of experiments, to optimize culture conditions to improve the yield and solubility of ΔNS1 in E. coli (Bae and Shoda 2005;Einsfeldt et al 2011;Emamipour et al 2019;Uhoraningoga et al 2018;Xie et al 2003). From the initial experiments performed using Erlenmeyer flasks, we changed to full-baffled flasks that improve the oxygen transfer rate, approaching the results to that obtained in bioreactors (Tunac 1989).…”

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

“…69 Despite being the most popular prokaryotic HE system, E. coli has many limitations that include inability to perform posttranslational modications, to fold eukaryotic proteins correctly which may lead to formation of inclusion bodies of insoluble proteins, or to recognize promoters and dismiss terminators and introns in lamentous fungal genes. 70 Codon bias, the inefficiency of tRNAs in E. coli to recognize certain codons in the foreign gene, could also reduce protein expression. The need to replace fungal promoters with host promoters, removal of terminators and introns, and codon optimization are laborious tasks which make E. coli a less suitable HE host, especially for expression of large fungal gene clusters.…”