Study of the covalent anchoring of proteins in the cell wall of Streptococcus thermophilus

Details

Supervisors

Soumillion, Patrice ; Hols, Pascal

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

Directed evolution is a growing research field with more and more tools and applications being developed. It consists of the generation of a library containing randomly mutated versions of a gene. The proteins encoded by each version of the gene are then selected or screened for a desired property and the genes coding for the protein of interest are recovered. The cycles of genetic diversification and screening/selection may be iterated for progressive improvement of the proteins. Many discoveries have been made in this field for industrial, research or even therapeutic applications. Those succesess have led directed evolution to be awarded in 2018 with the Nobel Prize in Chemistry. Half of the prize was awarded to George Smith and Sir Gregory P. Winter for their work on phage display. Phage display is a technology that allows the in vitro evolution of binding proteins such as antibodies by genetically fusing the proteins of the library to a coat protein of a bacteriophage. The displayed proteins are physically linked to their encoding gene through the phage particles and are available for in vitro selection by affinity capture. This technology has notably been key for the development of modern therapeutic antibodies. Display platforms have been developped with supramolecular entities, such as phage display, mRNA display and ribosome display, but also with cellular entities, such as yeast surface display, Escherichia coli surface display, etc. Display technologies are a very useful tool for directed evolution. As a matter of fact, the screening or selection of the protein variants that have the desired properties can be performed directly on the protein fused to the display platform. This makes it easier to retrieve and amplify the genes coding for the selected protein. The main objective of this master thesis is to develop a new display platform that could be useful in directed evolution experiments using Streptococcus thermophilus. Streptococcus thermophilus could be a good host for the displaying of recombinant proteins because of its highly efficient natural competence that facilitates the construction of highly diverse genetic libraries (up to 109 variants). As a gram-positive bacterium, the absence of an outer membrane should also greatly facilitate surface display. In order to build this display platform, the Streptococcus thermophilus LMD-9 strain will be used because the efficiency of its natural competence is extremely high, with up to 10% of total cells that can be transformed upon simple incubation of bacteria with XIP and linear DNA. Moreover, the LMD-9 strain encodes for a functional sortase A homolog that covalently anchors three different proteins on the cell wall. One of these proteins is a serine proteinase called PrtS. This protein comprises a sec-specific signal peptide that allows its translocation through the membrane, and an LPXTG motif for sortase-anchoring to the cell-wall. Our strategy for displaying recombinant proteins on the surface of Streptococcus thermophilus is to replace the sequence of the PrtS gene by the sequence of the protein of interest. The signal peptide and the anchoring motif of PrtS are retained and fused in frame with the sequence of the protein. As proof-of-concept, this display platform will be used to display the alpha-amylase from Bacillus licheniformis, a naturally secreted enzyme. A secondary objective will be to develop a display platform from which the protein can be detached from the bacteria with a reductive agent. To this aim, a bacterial extracellular intein-like (BIL) module will be inserted between the anchoring signal and the protein of interest. This module can auto-cleave itself from the precursor protein and leave a disulfide bond between both flanking polypeptides. A simple treatment of the cells with a reducing agent should then result in the release of the protein from the peptidoglycan. Such cleavable system may find useful applications in specific directed evolution campaigns that would take advantage of the controlled release of the displayed protein.