1. tarbiat modares university 2. tarbiat modares university
Abstract
Introduction
Staphylococcal protein a is a cell wall component which has the ability to bind to immunoglobulins of different animal species. protein a consists of three regions: signal sequence; five homologous igg binding domains and a cell-wall anchoring region. it can be used in various immunochemical assays including western blotting, immunohistochemistry, and elisa applications by conjugation with different reporter molecules, such as fluorescent dyes (fitc), enzyme markers (peroxidase, β-galactosidase, alkaline phosphatase), biotin, and colloidal gold.
the aim of this study is construction of protein a containing thiol groups for connecting to fluorescent dyes
Methods
Genomic dna of staphylococcus aureus, strain atcc6538, was extracted. cystein residue codons were introduced into the forward primer. then a fragment containing the igg binding region of protein a was amplified using pcr and cloned into the pet-26b(+). the resulting construct was transformed into e. coli strain bl21 and the bacterial cells were cultured in lb broth medium. the expression of the truncated of pa containing thiol group was induced by iptg after reaching a high od. the cells were harvested and the protein was isolated from the cell extraction.
Results
protein a containing thiol group finally was purified by nickle affinity chromatography.
Conclusion
purity of the obtained protein was confirmed by sds-page. the igg binding function of the protein was confirmed by igg antibody cross reaction
