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Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins

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Generation of Recombinant Antibodies against Orchardgrass Acidic nsLTP-Like Proteins

Gorica Rakleova, Ivanka Tsacheva, Mima Petkova, Ivelyn Pantchev, and Magdalena Tchorbadjieva*

Department of Biochemistry, Faculty of Biology, Sofia University, 8 Dragan Zankov str, 1164 Sofia, Bulgaria. Fax: 0 03 59 28 65 66 41. E-mail: magd@biofac.uni-sofia.bg

* Author for correspondence and reprint requests

Z. Naturforsch.63 c, 395Ð402 (2008); received November 20, 2007/January 9, 2008

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glome- rataL.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryo- genic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non- embryogenic lines were basic ones (pI 8Ð9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during so- matic embryogenesis. A naı¨ve phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellu- lose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressorE. colistrain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspen- sion cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteinsin vivois discussed.

Key words:Lipid Transfer Proteins, Somatic Embryogenesis, Recombinant Antibodies

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