Design of transcriptional fusions of stress sensitive promoters and GFP to monitor the overburden of Escherichia coli hosts during recombinant protein production

Abstract

Due to the lack of appropriate sensors for monitoring changes of Escherichia coli cells and the huge complexity of cellular systems, many of the present protein production processes are still far from optimal. Aiming at maximal exploitation of the host cell, enhanced knowledge of cellular reactions related to recombinant protein expression is required. Current methods like DNA microarrays and 2-D-electrophoresis enable the acquisition of transcriptional and translational activity shifts in stress situations like heat shock, general stress response, nutrient limitation, and stress caused by overexpression of heterologous proteins. However, these techniques and data processing are time consuming, therefore, the goal is to create new on-line systems such as stress promoter GFP fusions to monitor metabolic alterations. The fluorescence signal of expressed GFP can be measured by 2-D-multi-wavelength fluorescence spectroscopy, thereby allowing non-invasive on-line in vivo monitoring. Results of efficient stress monitoring approaches in ongoing protein production process are presented.

Soft sensor control of metabolic fluxes in a recombinant Escherichia coli fed-batch cultivation producing green fluorescence protein

“…A semi-synthetic medium described by Nemecek et al [29] was used for the pre-culture while a modified medium was used in the fed-batch cultivation. All reagents and chemicals were purchased from Merck, if not otherwise stated.…”

Anaerobic α-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae

“…This demands mechanisms of quality control and coordinated regulation, in order to avoid (or reduce) cellular stress that can result in reduced cell growth and protein secretion (1,2) or even apoptosis and cell death (3,4). Misfolded proteins are detected and removed via the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway (5), the ubiquitin-proteasome system (UPS) (5), the autophagy pathway (6), or the unfolded protein response (UPR) pathway (7).…”

Control of specific carbon dioxide production in a fed-batch culture producing recombinant protein using a soft sensor

“…Using the q CO 2 soft sensor for monitoring an E. coli fed-batch culture producing GFP Due to the favourable performance of the soft sensor we could use it for monitoring a typical fed-batch culture producing recombinant GFP (Albano et al, 1996;Nemecek et al, 2008). This expression system has previously been used by several researchers as a model for recombinant protein production.…”

“…A semi-synthetic medium (Nemecek et al, 2008) was used for the pre-culture while a modified medium was used in the fed-batch cultivation. All reagents and chemicals were purchased from Merck, if not otherwise stated.…”