Exchange of single amino acids at different positions of a recombinant protein affects metabolic burden in Escherichia coli

Abstract

BackgroundEscherichia coli is commonly used in academia and industry for expressing recombinant proteins because of its well-characterized molecular genetics and the availability of numerous expression vectors and strains. One important issue during recombinant protein production is the so-called ‘metabolic burden’: the material and energy normally reserved for microbial metabolism which is sapped from the bacterium to produce the recombinant protein. This material and energy drain harms biomass formation and modifies respiration. To the best of our knowledge, no research has investigated so far whether a single amino acid exchange in a recombinant protein affects the metabolic burden phenomenon. Thus, in this study, 15 E. coli BL21(DE3) clones expressing either the fusion tags, a recombinant wild type lipase, or 13 different lipase variants are investigated to quantitatively analyze the respective effects of single amino acid exchanges at different positions on respiration, biomass and protein production of each clone. Therefore, two small-scale online monitoring systems, namely a Respiration Activity MOnitoring System (RAMOS) and a microtiter plate based cultivation system (BioLector) are applied.ResultsUpon expression of all enzyme variants, strong variations were found in the Oxygen Transfer Rate (OTR), biomass and protein (lipase) production of the respective E. coli clones. Two distinct patterns of respiration behavior were observed and, so, the clones could be classified into two groups (Type A and B). Potential factors to explain these patterns were evaluated (e.g. plasmid copy number, inclusion body formation). However, no decisive factor could yet be identified. Five distinct cultivation phases could be determined from OTR curves which give real-time information about carbon source consumption, biomass and protein production. In general, it was found that the quantity of product increased with the duration of active respiration.ConclusionsThis work demonstrates that single amino acid exchanges in a recombinant protein influence the metabolic burden during protein production. The small-scale online monitoring devices RAMOS and BioLector enable the real-time detection of even smallest differences in respiration behavior, biomass and protein production in the E. coli clones investigated. Hence, this study underscores the importance of parallel online monitoring systems to unveil the relevance of single amino acid exchanges for the recombinant protein production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0191-y) contains supplementary material, which is available to authorized users.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

“…In their work, a correlation between OTR reflecting the metabolic state of the culture and recombinant protein production was observed. In addition, Rahmen et al confirmed these findings for E. coli BL21(DE3) clones expressing identical proteins only differing in a single amino acid or a single codon of the heterologous protein …”

“…Different frequently used T7‐based E. coli expression host strains were compared and evaluated regarding recombinant protein production of two model enzymes: a lipase A derived from B. subtilis and a 3‐hydroxybutyryl‐CoA oxidoreductase from T. thermophilus . The lipase is biochemically well characterized and under the chosen cultivation conditions predominantly expressed in inclusion bodies . It was fused to a flavin‐based fluorescent protein (FbFP) .…”

“…Both, soluble enzymes and enzymes in inclusion bodies are industrially relevant. Furthermore, both model enzymes have recently been investigated for recombinant protein production . Besides E. coli BL21(DE3), the standard host strain for recombinant protein production, four other strains of the B cell line, and two host strains derived from the K‐12 cell line are chosen for this comparative study.…”

Cellulolytic RoboLector – towards an automated high-throughput screening platform for recombinant cellulase expression

“…Glycerol is an additional energy source. It is consumed in parallel to lactose [33, 35, 36]. It was attempted to vary the time point of induction and the inducer concentration in order to influence the product formation [18, 36].…”

Determination of IPTG in Recombinant Human Growth Hormone with Ion Chromatography and Pulsed Electrochemical Detection

“…In the thioether group of IPTG (Figure 1), the sulfur atom creates a chemical bond which is non-hydrolysable by the cell, preventing the cell from degrading the inductant. IPTG has also been widely used in the recombinant processing of rhGH [7,8]. However, the toxicity of IPTG [9,10] restricted the usage of this promoter system, measuring its presence and concentration in the final products is of importance.…”