Creative BioMart to Present at
                        BIO-Europe Spring Creative BioMart to Present at AACR Annual Meeting|Apr. 5-10, 2024|Booth #2953

Escherichia coli Display Platform

E. coli, as a type of Gram-negative bacteria, is the most widely used cell display system in directed evolution. Since the first use for recombinant protein production, this system has been employed to display various proteins/peptides. With the power of high-throughput technologies, such as magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques, E. coli display can be used in many applications of protein evolution, using the creation of novel vaccines, the optimization of enzyme substrates and improved affinity of a ligand for its target protein as examples.

The target proteins are often displayed on the surface of the E. coli, namely cell surface display, rather than in the cytoplasm of the cell. To achieve this strategy, a carrier protein (anchoring motif) is required. Since E. coli possess a complex cell envelope structure, there are plenty surface-anchoring motifs that can be used for display. In general, different strategies based on different surface-anchoring motifs include: i) insertion of the target sequences into the surface exposed outer membrane proteins; ii) insertion of target sequences into a protein forming part of a cell surface structure such as a flagellum; or iii) the fusion of target sequences to the N-terminus of lipoproteins. About a decade ago, E. coli display became a powerful tool for directed evolution of membrane proteins, which was expressed in the inner membrane of E. coli.

E. coli is also applied in in vivo mutagenesis approaches. An early example was carried out by the Janniere group, who used a low fidelity DNA Pol I mutant that preferentially amplified a plasmid over the bacterial chromosome in E. coli. This idea has been extended to targeted mutagenesis in Saccharomyces cerevisiae by Chang Liu and coworkers.

Creative BioMart is an expert in directed evolution using E. coli display system, as well as other Gram-negative bacteria systems such as Proteus mirabilis. Based on years of experience, we have developed various strategies to display target proteins/peptides on the surface of E. coli that is suitable for a followed high-throughput screening. We have successfully engineered various proteins for different properties. With advanced knowledge and instruments, we offer one-stop services of protein engineering:

  • Selection of coat protein, signaling molecules and strains for target protein/peptide.
  • Validated expression and high-throughput screening.
  • Selected/screened clone amplification and sequencing.
  • Recombinant protein expression, purification and characterization.

Services Features

  • Libraries with large diversity sizes (approximately 108-10).
  • In vitro and in vivo mutagenesis for genetic diversity.
  • Other Gram-negative bacterial systems available.
  • High-throughput technologies: MACS and FACS.
  • Various desired functions as target.

References:

  1. Sarkar, C.A., Dodevski, I., Kenig, M., Dudli, S., Mohr, A., Hermans, E., Plückthun, A. (2008) Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proceedings of the National Academy of Sciences of the United States of America. 105: 14808-14813.
  2. d'Oelsnitz, S., Ellington, A. (2018) Continuous directed evolution for strain and protein engineering. Current Opinion in Biotechnology. 53: 158-163.

e. coli cell surface display in directed evolution. Figure 1 Surface display systems developed in Gram-negative bacteria.
(Trends in Biotechnology 2003)

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