Expression Vectors for the Construction of Hybrid Ty-VLPs
The synthesis of recombinant proteins or protein domains in microbial, insect, or mammalian systems is now commonplace in molecular biology laboratories. The gene or gene fragment encoding the protein of interest is inserted into a specialized expression vector, flanked by efficient transcription and translation control sequences. The expression vector is then inserted into recipient cells and expression of the protein induced. The expressed protein then has to be purified from other cellular or medium components. Purification can be facilitated by expressing the recombinant protein as a fusion with a carrier protein that assembles into particulate structures. This approach has been developed using a protein encoded by the yeast retrotransposon Ty, which self-assembles into virus-like particles (VLPs) (1,2). Additional protein coding sequences can be fused to the carrier protein gene and expressed in yeast to produce hybrid Ty-VLPs (3,4). The physical characteristics of the VLPs have been exploited to produce a rapid purification procedure that is essentially generic for any hybrid construction. Hybrid VLPs can be used in many laboratory applications (see elsewhere in this vol), including the production of polyclonal and monoclonal antibodies, structure/function analyses, the detection of important antigenic determinants, and epitope mapping of monoclonal antibodies.
KeywordsKlenow Fragment Calf Intestinal Alkaline Phosphatase Ethidium Bromide Solution YEPD Medium Sorvall SS34 Rotor
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