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The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

ABSTRACT: Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl ?-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

SUBMITTER: Wurm DJ

PROVIDER: S-EPMC5035661 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

Wurm David Johannes DJ Veiter Lukas L Ulonska Sophia S Eggenreich Britta B Herwig Christoph C Spadiut Oliver O

Applied microbiology and biotechnology 20160527 20

Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-D-1-thiogalactop ...[more]

PMID: 27229726

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Project description:The malignancy potential is correlated with mechanical deformability of the cancer cells. Therefore, mechanical properties of individual patient cells can contribute essential knowledge in cancer diagnostics and in precision therapies. However, clinical applications are limited by lack of current knowledge and of reliable and robust cell mechanical tests. In a comprehensive study, we found a Triangular Correlation (TrC) between cell deformability, phagocytic like capacity, and cancer aggressiveness. A significant application of the TrC is that phagocytic measurements can be a surrogate marker for detecting malignancy of cancer cells based on mechanical properties. We found that the uptake of inert sub-micron and micro-beads was massively higher in cancer cells compared with non-cancerous cells. Moreover, cells with a higher malignancy potential had greater uptake capacity. Importantly, in a reciprocal approach, we sorted either human bladder cancer cells or melanoma cells into subpopulations, solely based on their phagocytic capacity. The more phagocytic subpopulations showed elevated phenotypes of cancer aggressiveness ex vivo and in vivo. The uptake potential was found to be an imprinted feature preserved genetically and enriched over the sorting cycles. A gene expression profile revealed differences in gene sets associated with regulation of cell-cell and extracellular matrix adhesions and epithelial-to-mesenchymal transition. Chromatin immunoprecipitation sequencing showed that differences in gene expression were reflected also at the chromatin level. Importantly, in all cases studied here, enhanced phagocytic ability and elevated aggressiveness phenotypes were correlated with greater cell deformability, providing the mechanical link of the TrC. A computational model supported the notion that the uptake capacity can be a marker for malignancy mediated by cell mechanics. Our multidisciplinary approach, which involved complementary directions of study in several types of cancer cells, provides the proof of concept that phagocytic measurements can be applied for cancer diagnostics and precision medicine.

Dataset Information

The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

Publications

The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

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