Vascular proteomics and subtractive antibody expression cloning.
The cloning of genes expressing proteins that are differentially expressed in the organ microvasculature has the potential to address a variety of problems ranging from the analysis of disease pathogenesis to drug targeting for particular tissues. This study describes a methodology designed to analyze differential protein expression in the brain microvasculature. The method can be applied to other organs and is particularly suited to the cloning of cDNAs encoding membrane proteins. The technology merges a tissue-specific polyclonal antiserum with a cDNA library expression cloning system. The tissue-specific antiserum is subtracted with protein extracts from control tissues to remove those antibodies that recognize common antigenic proteins. Then, the depleted antiserum is used to expression clone tissue-specific proteins from a cDNA library expressed in mammalian cells. The methodology was evaluated with a rabbit polyclonal antiserum prepared against purified bovine brain capillaries. The antiserum was absorbed with acetone powders of liver and kidney and then used to screen a bovine brain capillary cDNA library in COS cells. The initial clone detected with this expression methodology was the Lutheran membrane glycoprotein, which is specifically expressed at the brain microvasculature compared with liver and kidney tissues. This subtractive expression cloning methodology provides a new approach to "vascular proteomics" and to the detection of proteins specifically expressed at the microvasculature, including membrane proteins.
Shusta
Eric V
EV
Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024, USA.
Boado
Ruben J
RJ
Pardridge
William M
WM
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply*; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004730:Endothelium, Vascular; D015870:Gene Expression; D015723:Gene Library; D007124:Immunoenzyme Techniques; D008188:Lutheran Blood-Group System / Q000032:analysis*; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry*; D015203:Reproducibility of Results; D013382:Subtraction Technique; D014162:Transfection
D004274:DNA, Recombinant; D008188:Lutheran Blood-Group System; D020543:Proteome