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A scalable screening of E. coli strains for recombinant protein expression.

ABSTRACT: Structural biology projects are highly dependent on the large-scale expression of soluble protein and, for this purpose, heterologous expression using bacteria or yeast as host systems is usually employed. In this scenario, some of the parameters to be optimized include (i) those related to the protein construct, such as the use of a fusion protein, the choice of an N-terminus fusion/tag or a C-terminus fusion/tag; (ii) those related to the expression stage, such as the concentration and selection of inducer agent and temperature expression and (iii) the choice of the host system, which includes the selection of a prokaryotic or eukaryotic cell and the adoption of a strain. The optimization of some of the parameters related to protein expression, stage (ii), is straightforward. On the other hand, the determination of the most suitable parameters related to protein construction requires a new cycle of gene cloning, while the optimization of the host cell is less straightforward. Here, we evaluated a scalable approach for the screening of host cells for protein expression in a structural biology pipeline. We evaluated four Escherichia coli strains looking for the best yield of soluble heterologous protein expression using the same strategy for protein construction and gene cloning and comparing it to our standard strain, Rosetta 2 (DE3). Using a liquid handling device (robot), E. coli pT-GroE, Lemo21(DE3), Arctic Express (DE3), and Rosetta Gami 2 (DE3) strains were screened for the maximal yield of soluble heterologous protein recovery. For the genes used in this experiment, the Arctic Express (DE3) strain resulted in better yields of soluble heterologous proteins. We propose that screening of host cell/strain is feasible, even for smaller laboratories and the experiment as proposed can easily be scalable to a high-throughput approach.

SUBMITTER: Morao LG

PROVIDER: S-EPMC9312941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale.The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell culture will also be useful for applications such as the generation of receptor protein microarrays.

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A scalable screening of E. coli strains for recombinant protein expression.

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