Transmembrane Protein Center:


The use of PIPE cloning at TMPC

Transmembrane Protein Center

Polymerase Incomplete Primer Extension (PIPE) cloning is a ligase free cloning technology developed at the Joint Center for Structural Genomics (1,2). We use PIPE cloning extensively for the initial creation of expression clones. When the clones are made PIPE technology is also used to modify them by deleting or exchanging tag or linker sequences, creating truncation variants, and making point mutations.

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Variations in linker region of MBP-S1R fusion protein dramatically influence the yield of highly purified, active membrane receptor

Transmembrane Protein Center

Different lengths of linkers and expression host cell lines were tested in order to improve the yield of the MBP-S1R fusion protein. First attempts to express and purify MBP-SR1 replicate published results of our collaborator Professor A. Ruoho, who reported ~0.3 mg of protein/liter of cell culture. By decreasing the length of the amino acid linker, we increased the expression by a factor of 10 with the best-performing 3 alanine (AAA) and by a factor of 12 with 4 alanine (AAAA) linker construct.

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Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

Transmembrane Protein Center

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed.

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