Restriction digests of the clone give the following sizes (kb): NotI--5.0; EcoRI/PstI--4.0, 1.0; BamHI/EcoRV--3.2, 1.8. Cloning into this vector requires amplification of the gene using oligonucleotides prepared as in the reference and encoding the first 4 amino acids of a thrombin recognition sequence. Expression vector for rapid purification of fusion proteins that contain no amino terminal extensions after thrombin cleavage. The amino acid after the initiator methionine must be charged. The glutathione S-transferase (GST) fusion protein can be purified by glutathione affinity chromatography, and the desired polypeptide released from the fusion product by thrombin. Constructed from pGEX-1 by inserting an oligonucleotide at the BamHI site which encodes the glycine "kinker" and a NotI site. |
