Abstract
Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.
Highlights
The expression of recombinant proteins fused with signal peptides to sort and direct them to different parts of the bacterial cell surface is called surface display which has many applications such as peptide libraries screening, whole-cell bioconversion, live vaccine production, and whole-cell metal adsorbents [20].The surface display is a suitable alternative for immobilizing recombinant proteins and enzymes on various polymeric supports and living cells because, unlike traditional physical and chemical immobilization approaches that require various steps, it is biocompatible and not time-consuming or challenging to achieve [25].There are several ways to efficiently display heterologous polypeptides on the surface of the bacterial cells
The plasmid constructs were first transformed in E. coli as a cloning host and into B. subtilis as an expression host (Additional file 1: Fig. S3, S4)
Bs-NC1 is a recombinant B. subtilis transformed with pNC1 [a recombinant pHY plasmid containing spa gene and cell wall sorting motif (CWM) but lacks the enzyme sortase]
Summary
There are several ways to efficiently display heterologous polypeptides on the surface of the bacterial cells. Various surface proteins such as outer membrane lipoproteins including LamB, OmpA, and OmpF in gramnegative bacteria are used as fusion partners to express bacterial and viral antigens [3, 8, 22, 37]. Gram-positive bacteria are more rigid because they have a much thicker peptidoglycan layer than that of gram-negative bacteria. They lack a lipid outer membrane envelope, which simplifies the extracellular secretion of heterologous proteins [32]. One of the most attractive strategies used for superficial display of homologous and heterologous proteins by a number of gram-positive bacteria is sortase-mediated immobilization
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