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Generation of a phage‐display library of single‐domain camelid V H H antibodies directed against C hlamydomonas reinhardtii antigens, and characterization of V H H s binding cell‐surface antigens
Author(s) -
Jiang Wenzhi,
Rosenberg Julian N.,
Wauchope Akelia D.,
Tremblay Jacqueline M.,
Shoemaker Charles B.,
Weeks Donald P.,
Oyler George A.
Publication year - 2013
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12316
Subject(s) - chlamydomonas reinhardtii , antigen , biology , microbiology and biotechnology , phage display , antibody , western blot , epitope , gene , biochemistry , genetics , mutant
Summary Single‐domain antibodies (sd A bs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid ( L ama pacos ) heavy chain‐only variable V H domain ( V H H ) libraries against antigens in total cell lysates from C hlamydomonas reinhardtii . The sd A bs in the sera from immunized animals and V H H antibody domains isolated from the library show specificity to C . reinhardtii and lack of reactivity to antigens from four other algae: C hlorella variabilis , C occomyxa subellipsoidea , N annochloropsis oceanica and T halassiosira pseudonana . Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V H H region contained at least 10 6 individual transformants, and thus should represent a wide range of C . reinhardtii antigens. The utility of the phage library was demonstrated by using live C . reinhardtii cells to pan for V H H clones with specific recognition of cell‐surface epitopes. The lead candidate V H H clones (designated B 11 and H 10) bound to C . reinhardtii with EC 50 values ≤0.5 n m . Treatment of cells with V H H B 11 fused to the m C herry or green fluorescent proteins allowed brilliant and specific staining of the C . reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V H H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.