A VECTOR FOR DIRECTIONAL CLONING AND EXPRESSION OF POLYMERASE CHAIN-REACTION PRODUCTS IN ESCHERICHIA-COLI

This paper describes the construction of a modified vector for the cloning and expression of protein-encoding genes in Escherichia coli. The vector, pfXblue, is derived from the system originally developed by Nagai and Thogerson [Nature 309 (1984) 810-812], but contains a modified multiple cloning site (MCS) from M13mp18 to allow directional insertion of foreign coding sequences. The MCS is located within the M13mp18 lacZ' gene and thus allows blue/white screening of colonies for inserts. The inserted gene is expressed as a fusion protein, which, when cleaved by the coagulation factor Xa protease, yields the mature product. This vector was successfully used for the production of a mitochondrial [FE2-S2]ferredoxin using polymerase chain reaction products generated from a chick kidney cDNA library.