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 Thøgerson [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 [2Fe-2S]ferredoxin using polymerase chain reaction products generated from a chick kidney cDNA library.
Brandt
M E
ME
Department of Physiology and Biophysics, University of California, Irvine 92717.
Gabrik
A H
AH
Vickery
L E
LE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa / Q000378:metabolism; D005288:Ferredoxins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
D005288:Ferredoxins; D011993:Recombinant Fusion Proteins; D015951:Factor Xa