Journal: bioRxiv
Article Title: A major histocompatibility complex (MHC) class II molecule that binds the same viral pathogen peptide with both nonamer and decamer core sequences for presentation to T cells
doi: 10.1101/2025.10.27.684722
Figure Lengend Snippet: BL2*21:01 linked to the MDV pp38 peptide was expressed in insect cells and purified, and has a similar thermostability as other chicken class II molecules bound to MDV peptides. A. Cartoon showing full-length construct, with scissors indicating the potential cleavage sites of endoproteinase Glu-C (Endoproteinase V8). B. UV traces of FPLC size-exclusion chromatography (SEC) using Superdex S200 column, before (blue) and after (black) being subjected to Endoproteinase V8 cleavage at 37°C overnight to remove C-terminal tags and dimerization domains. C. SDS-PAGE followed by Coomassie blue staining after initial nickel column purification (Input) and SEC fractions comprising Peak1 (blue trace of profile in A). Standard protein markers with indicated molecular masses; blue arrows indicate position of α- and β-chains. D. SDS-PAGE followed by Coomassie blue staining of endoproteinase V8 on its own, Peak1 (blue trace of profile in A), and SEC fractions from Single Peak (black trace). Standard makers and blue arrows is in C, black arrows indicate the position of the endoproteinase V8 cleaved α- and β-chains. E. Thermal denaturation curves for BL2*02:01 molecules expressed with MDV peptides from glycoprotein H (gH, with melting temperature 69°C), glycoprotein E (gE, 75°C), and unique long gene 43 tegument protein (UL43, 78°C), compared to BL2*021:01 molecule expressed with MDV pp38 peptide.
Article Snippet: Sf9 insect suspension cells ( Spodoptera frugiperda female ovarian cell line, ATCC CRL-1711) were used for production of baculovirus, and High Five insect suspension cells ( Trichoplusia ni female ovarian cell line, GIBCO B85502) were used for protein production, both grown in Insect-XPRESS Protein-free Insect Cell Medium (Lonza, BE12-730Q) supplemented with L-glutamine (to 1%), 50 U/ml penicillin, and 50 μg/ml streptomycin at 27°C with shaking at 135 rpm.
Techniques: Purification, Construct, Size-exclusion Chromatography, SDS Page, Staining, Nickel Column
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