Cloning and expression of functional equistatin.
Equistatin is a 199-residue protein composed of three thyroglobulin type-1 domains. It strongly inhibits cysteine proteinases as well as the aspartic proteinase cathepsin D. In order to initiate structure-function studies by protein engineering, a cDNA library from sea anemone, Actinia equina, was screened. A positive clone of 888 nucleotides was shown to encode a protein of 231 amino acids, including the signal sequence. The mature protein region was amplified by PCR, cloned into the pET22b(+)cas expression vector and expressed in Escherichia coli. Isolation of active recombinant equistatin required only one purification step, the His-tag affinity column. The protein displays physical and inhibitory properties closely similar to the native inhibitor.
Galesa
K
K
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia.
Strukelj
B
B
Bavec
S
S
Turk
V
V
Lenarcic
B
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; C106356:equistatin