Development and characterization of screening platforms in microtiter plates for the production of recombinant proteins

RWTH Publications (RWTH Aachen)

Abstract

Recombinant proteins play a key role in many sectors of the bioeconomy, predominantly in pharmaceutical and industrial biotechnology. Regardless of the final application, highly efficient screening and production of recombinant proteins is essential for economic feasibility. One cell-free expression platform that meets these requirements is the Almost Living Cell-free Expression (ALiCE) system, which is based on tobacco BY-2 cells. The first part of this work focuses on gaining a deeper understanding of oxygen demand, hydromechanical stress and protein formation in this cell-free expression system. Online monitoring was established in a combined µRAMOS-BioLector device to investigate the correlation of oxygen transfer, eYFP production and NADH levels during the ALiCE reaction. By varying the maximum oxygen transfer capacity, it was revealed that protein formation is strongly dependent on oxygen availability and that the eYFP production rate decreases with decreasing oxygen availability. Furthermore, experiments with inhibited enzymes of the mitochondrial respiratory chain revealed that a functional alternative oxidase, complex III and IV are essential for maximizing protein titer within ALiCE. Furthermore, the impact of hydromechanical stress was studied by varying the volumetric power input. The detrimental effects observed at an increased volumetric power input of 0.51 kW/m3 were eliminated by adding the amphiphilic compound A to the ALiCE reaction. Compound A was proven to be the best candidate out of 21 tested amphiphilic compounds. This finding enabled the scale-up of ALiCE to shake flasks and a considerable expansion of the operational window. Besides cell-free systems, also cell-based platforms are widely used to produce recombinant proteins from the screening phase to manufacturing. In this context, yeast-based platforms are common, due to their secretory capabilities and high yields. In the second part of this work, recombinant phytase production with the yeast Komagataella phaffii was optimized in terms of increasing the efficiency of the primary screening. The objective was to predict high recombinant protein producer strains as well as phytase variants with high volumetric activity, based on the respiratory activity of the host, K. phaffii. Online monitoring of the oxygen transfer rates in 96-well plates enabled the evaluation of the total protein concentration and the volumetric phytase activity already during the expression. Using these results, also the specific activity can be calculated. In the future, primary screening experiments of large enzyme mutant libraries can be conducted without offline activity assays, to identify promising candidates.

Authors 1

1. Sarah Luise Straaten corresponding Aachen

   RWTH Aachen University

   Affiliation as printed

   RWTH Aachen