Method of high-throughput sorting of high expression cell and cells sorted therefrom
AssigneeTaipei Medical University
InventorsKuo-Hsiang Chuang, Tian-Lu Cheng, I-Shiuan Chiang, Yuan-Chin Hsieh
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Abstract
The invention provides a method of high-throughput sorting of high expression protein-producing cell, which utilizes linking a protein and a transmembrane domain with a self-processing cleavage site and regulating the secretion of the protein or expression of protein on the cell membrane by adding self-processing cleavage enzyme inhibitor. Then, the high expression cell line can be high-throughput sorted by a detection technique. The invention also provides a recombinant nucleotide sequence and a vector used in the method and a cell sorted by the method.
Claims14
A method of high-throughput sorting of protein-expression cells, comprising the following steps:
- (a) constructing a vector comprising a recombinant nucleotide sequence with a detectable label, which comprises, in 5′ to 3′ direction, a first sequence encoding a protein or a polypeptide of interest, a second sequence encoding a self-processing cleavage site and a third sequence encoding an extracellular-transmembrane domain, wherein the second sequence is located between the first sequence and the third sequence, and wherein the detectable label is located at the first sequence or the third sequence;
- (b) transfecting a cell with the vector of (a);
- (c) adding a self-processing cleavage enzyme inhibitor so that the recombinant protein encoded by the recombinant sequence of the vector of (a) can be transiently anchored on a cell surface;
- and
- (d) determining the detectable label to sort the recombinant protein expressing cells that express anchored recombinant protein on the cell surface to obtain protein-expression cells.
Dependencies are inferred from the claim text.