Gene fusions with human carbonic anhydrase II for efficient expression and rapid single-step recovery of recombinant proteins: expression of the Escherichia coli F1-ATPase epsilon subunit.
A new expression vector was constructed which allows the overproduction in Escherichia coli of tripartite proteins consisting of human carbonic anhydrase isozyme II (hCAII), a peptide linker containing an enterokinase cleavage site, and a target protein of interest. Carbonic anhydrase is soluble and stable in E. coli and serves as a highly specific purification tag in the recovery of the fusion protein by a single affinity chromatography step. The enterokinase cleavage site was engineered into the construct to allow accurate and efficient release of the target protein. To demonstrate the practical value of this vector, the E. coli F1-ATPase epsilon subunit was expressed as a fusion with hCAII. After a single purification step, biologically active recombinant E. coli F1-ATPase epsilon subunit was recovered following proteolytic removal of the hCAII moiety.
Protein Expression Laboratory, University of Florida, Gainesville.
G
G
Van Heeke
R
R
Shaw
R
R
Schnizer
J M
JM
Couton
S M
SM
Schuster
F W
FW
Wagner
D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000302:isolation & purification; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D004247:DNA; D006180:Proton-Translocating ATPases; D002256:Carbonic Anhydrases