Special Cell-based Display Platform

Besides commonly used hosts, such as E. coli, yeast and phage, there are other cells that can also be used for protein display, although not widely employed. Gram-positive bacteria display method, for example, is playing a special role in directed evolution. Because of the rigid structure of their cell walls, this system is suitable for whole-cell catalysts and whole-cell adsorbents. Also, taking advantages of the inert properties of endospores, which usually occurs in Gram-positive bacteria, several directed evolution experiments have been established for improved properties under harsh conditions. Bacillus and Staphylococcus strains have been used most often in this technology.

The principle behind Gram-positive bacteria display technology is analogous to other cell display methods, with the recombinant protein anchored on the surface and the encoding DNA encapsulated inside the cell. Using B. subtilis as an example, proteins can be anchored to the plasma membrane through fusion to PrsA, but lysozyme treatment is necessary to expose this cellulase to the external environment. More conventionally, proteins are cell surface displayed either through noncovalent interactions with cell wall peptidoglycan by protein fusion to the LysM domain, or through covalent attachment to cell wall peptidoglycan by protein fusion to a cell wall sorting signal containing the LPXTG motif, which is processed by sortase transpeptidase.

B. subtilis sporulates to produce a highly stress‐resistant, dormant spore cell that can be used to overcome some specific problems in traditional protein display. As the most resistant form of life on earth and remain viable under extreme chemical and physical conditions, endospores can be used to evolve proteins to possess extreme properties. Also, protein folding problems associated with the target protein traveling through cell membranes is eliminated, which is due to the natural sporulation process. The spore-display system is based on the construction of gene fusions between heterologous DNA and a B. subtilis gene coding for an endospore coat protein such as CotG, CotB and CotC.

Creative BioMart has established a platform of Gram-positive bacteria display to meet special needs from our customers. With advanced knowledge and instruments, we offer one-stop services of protein engineering:

• Protein display on surface of Gram-positive bacteria: B. subtilis, lactic acid bacteria (LAB), C. glutamicum, ...
• Validated high-throughput screening.
• Selected/screened clone amplification and sequencing.
• Recombinant protein expression, purification and characterization.

Features

• Libraries with large diversity sizes (approximately 10^8-10).
• Featured B. subtilis display platform.
• Screening under harsh condition for extreme properties.

Reference:

1. Huang, G.L. and Clubb, R.T. (2016) Progress towards engineering microbial surfaces to degrade biomass, in Biomass, J. Tumuluru, Editor.

Figure 1 Surface display systems developed in Gram-positive bacteria.
(Huang, G.L. and Clubb, R.T. 2016)