Restriction digests of the clone give the following sizes (kb): SacII--5.1; PstI--5.1; SalI--5.1; PstI/BamHI--4.0, 0.98. Vector is covered by a U.S. patent application and is distributed only for non-commercial use. The carrier protein can be removed by thrombin proteolysis. Preparation of the vector for cloning includes linearization with SacII, gel purification of the linearized vector, and treatment with T4 DNA polymerase in the presence of dATP. Target sequences for cloning are prepared by PCR and do not require restriction enzyme digestion. Annealing the vector and amplification product forms a gapped duplex molecule that can be used directly to transform bacteria without ligation. Ligation-independent expression vector for constructing fusion proteins with a carrier polypeptide (glutathione S-transferase) which allows single-step affinity purification. The forward primer should contain 15 nt complementary to nt 5' to the SacII site of the vector [(5'-3')(GGCCTGGTTCCGCGG)] followed by 12-15 nt corresponding to the target sequence (nt encoding the N-terminal 4-5 aa of the protein). The reverse primer should contain 14 nt complementary to nt 3' to the SacII site of the vector [(5'-3') (CTGCGCCTCGCTCC)] followed by 12 nt complementary to the 3' end of the target sequence. Both primers should contain a dAMP residue near the sequence complementary to the vector to terminate the exonucleolytic activity of the subsequent T4 DNA polymerase treatment. Design of PCR primers permits cloning in any reading frame. The order of the major features in this plasmid is: pMB1 ori - lacIq - lacZ' - tac - GST/SalI/SacII/thrombin cleavage site/BamHI - ampR. |
