Matching Items (1)
Description
Our goal was to design a method to express soluble folded major histocompatibility complex (MHC) proteins using human cell line HeLa lysate with the novel 1-Step Human In Vitro Protein Expression by Thermo Scientific in the presence of β2 microglobulin (β2m) and antigenic peptide.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
We confirmed that the soluble protein MHC-A2.1 could be successfully attached to the Luminex magnetic beads and detected using the primary antibody anti-GST and the detection antibody goat mAb mouse PE. The average net MFI of the attached pA2.1-bead complex was 8182. Biotinylated A2.1 MHC complexes pre-folded with β2m and FLU M1 peptide (A2.1 monomers) were also successfully attached to Luminex magnetic beads and detected with BB7.2. The average net MFI of the detected A2.1 monmer-bead complexes was 318. The protein MHC complexes were multimerized on magnetic beads to create MHC tetramers and detected with BB7.2, PE labeled monoclonal antibody, via median fluorescent intensity with the Luminex platform. Varying protein, β2 microglobulin (β2m), and peptide concentrations were tested in a number of MHC-A2.1 protein refolding trials. Different antigenic peptides and attachment methods were also tested. However, none of the MHC-A2.1 protein folding and capture trials were successful. Although MHC-A2.1 complexes and recombinant MHC molecules could be attached to Luminex magnetic beads and be detected by Luminex arrays, soluble protein A2.1 could not be successfully expressed, refolded, captured onto Luminex beads, and detected. All refolding trials resulted in a net MFI of <25. The failed refolding and capture trials of A2.1 lead to the conclusion that human cell line HeLa lysate cannot be used to properly fold MHC molecules. However, efforts to refold the complexes onto Luminex magnetic beads are ongoing. We are also using the baculovirus expression system to refold soluble A2.1 lysate onto peptide-bead complexes.
ContributorsChang, Peter S (Author) / Anderson, Karen (Thesis director) / Chang, Yung (Committee member) / Sundaresan, Krishna (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05