Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system.
The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.
Szweda
P
P
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, 80-952 Gdańsk, Poland.
Pladzyk
R
R
Kotlowski
R
R
Kur
J
J
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D002686:Chitin; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008248:Lysostaphin / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013210:Staphylococcus / Q000378:metabolism
D000345:Affinity Labels; D011993:Recombinant Fusion Proteins; D002686:Chitin; D008248:Lysostaphin