Functional inhibition of transitory proteins by intrabody-mediated retention in the endoplasmatic reticulum.
Intrabodies are recombinantly expressed intracellular antibody fragments that can be used to specifically bind and inhibit the function of cellular proteins of interest. Intrabodies can be targeted to various cell compartments by attaching an appropriate localization peptide sequence to them. An efficient strategy with a high success rate is to anchor intrabodies in the endoplasmatic reticulum where they can inhibit transitory target proteins by binding and preventing them to reach their site of action. Intrabodies can be assembled from antibody gene fragments from various sources into dedicated expression vectors. Conventionally, antibody cDNA sequences are derived from selected hybridoma cell clones that express antibodies with the desired specificity. Alternatively, appropriate clones can be isolated by affinity selection from an antibody in vitro display library. Here an evaluation of endoplasmatic reticulum targeted intrabodies with respect to other knockdown approaches is given and the characteristics of various intrabody expression vectors are discussed. A step by step protocol is provided that was repeatedly used to construct intrabodies derived from diverse antibody isotypes producing hybridoma cell clones. The inactivation of the cell surface receptor neural cell adhesion molecule (NCAM) by a highly efficacious novel endoplasmatic reticulum-anchored intrabody is demonstrated.
Department of Gene Regulation and Differentiation-RDIF, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Thomas
T
Böldicke
Stefan
S
Somplatzki
Galina
G
Sergeev
Peter P
PP
Mueller
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001483:Base Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019006:Neural Cell Adhesion Molecules / Q000037:antagonists & inhibitors; D019151:Peptide Library; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D016372:RNA, Antisense
D000906:Antibodies; D019006:Neural Cell Adhesion Molecules; D019151:Peptide Library; D011506:Proteins; D016372:RNA, Antisense