In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers.
Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP (BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.
Department of Surgery, Washington University School of Medicine, MO 63110-1093, USA.
Junbao
J
Yang
Andrés
A
Jaramillo
Ruili
R
Shi
William W
WW
Kwok
T
T
Mohanakumar
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019921:Biotinylation / Q000379:methods*; D015153:Blotting, Western; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004331:Drosophila melanogaster / Q000166:cytology; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D015802:HLA-DR2 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008213:Lymphocyte Activation / Q000276:immunology; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection / Q000379:methods
D029968:Escherichia coli Proteins; D015802:HLA-DR2 Antigen; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D000949:Histocompatibility Antigens Class II; D004676:Myelin Basic Protein; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; C086595:influenza hemagglutinin (306-318); C107908:myelin basic protein 85-99; D001710:Biotin; D019809:Streptavidin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli