Team:TMMU China/Project Results/Project3
Surface display system
Background
The anchoring of proteins to the cell surface of lactic acid bacteria (LAB) using genetic techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications, including vaccine development, whole cell catalysis, bioremediation and etc. In iGEM, the 2015 Concordia University-Canada team tried but failed to develop a protein scaffold surface display system. Development of a surface display system in L. lactis will make it a better chassis in synthetic biology.
To build a surface display system, the first thing should be considered is the cell wall anchoring motif. The most exploited anchoring regions are those with the LPXTG motifs that bind the proteins in a covalent way to the cell wall. However, to achieve versatile application, it is better to use anchoring domains that interact with the cell wall in a non-covalent way. The major autolysin of L. lactis, the cell wall hydrolase AcmA contains 3 tandem arranged LysM motifs and separated by stretches of 21 to 31 amino acids, this region is collectively termed as the cA anchoring domain. The cA domain can be fused to the N- and C- terminus of functional proteins, and can bind proteins to the cell walls of a broad range of gram-positive bacteria. Interaction of the cA anchoring domain with the cell wall is less strong than that of the LPXTG type cell wall anchoring motif. The interaction between cA and the cell wall is non-covalent. The cA domain could also be used to immobilize soluble enzymes in an active form on the cell surface of L. lactis. Like AcmA, these chimaeric enzymes could bind to L. lactis cells when added from the outside. Thus, cA fusion proteins produced using other expression systems can also be attached to the cell wall of L. lactis.
To get the protein of interest anchored to the surface of L. lactis, it should be secreted out of the cell first. The solution is a signal peptide from the Usp45 protein which is secreted out of L. lactis very efficiently. Fusion of the signal peptide of Usp45 to a couple of proteins resulted in an efficient way of secreting the coded proteins into the media. The fusion protein will meet a chaperone protein, SecB, which bring the fusion protein to a protein secretion apparatus. This apparatus cleaves the signal peptide and frees the proteins from the cell.
Figure3.1:The surface display system
Design
The protein of interest is fused to the cA domain with the USP45 signal peptide, driven by the PnisZ promoter and followed by the nisin resistant gene nsr. To demonstrate the utility of the protein surface display, here we took the β-galactosidase protein as a proof of concept.
Figure3.2:Design of the surface display system
Result
To demonstrate whether cA could indeed anchor the protein of interest at the surface of L. lactis, the pLacZ-Cytosol and pLacZ-Surface plasmids were introduced into NZ9000 respectively. As stated above, the cA domain is derived from the AcmA protein, which is an autolysin of L. latis that cleaves the peptidoglycan to release the duplicated bacteria. Since the substrate peptidoglycan is now occupied by the cA-β-galactosidase fusion proteins, the AcmA autolysin activity is hindered, thus cell separation will be interfered. Indeed, we found that under microscopic, the NZ-Surface cells were poorly separated compared to the NZ-Cytosol strain. Further more, using a polyclonal antibody against β-galactosidase, we found that about 70% of the β-galactosidase protein is present at the cell wall, while the other 30% protein is present in the cytoplasm, perhaps due to the inefficient secretion process. In contrast, all theβ-galactosidase proteins were present in the cytoplasm in the NZ-cytosol strain.
Figure3.3:The protein surface display system for L. lactis. A). Microscopic picture of the NZ-Cytosol and NZ-surface. The LysM domains of cA can compete with the autolysins of L. lactis, thus the NZ-surface strain is poorly separated after nisin induction. B). Western blot confirmation of cell wall anchored proteins using LacZ as an example. Lane1, NZ9000 whole proteins. Lane 2, NZ-Surface cytosolic proteins. Lane 3, NZ-Surface cell wall proteins. Lane 4, NZ-Surface membrane proteins. Lane 5, cytosolic proteins of NZ-cytosol. Lane 6, cell wall proteins of NZ-cytosol. Lane 7, membrane proteins of NZ-cytosol.
