Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II).
Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0 degrees C to 70 degrees C. The work described here can also have implications for understanding protein stability in vitro and in vivo.
Andberg
Martina
M
VTT Technical Research Centre of Finland, Espoo FIN-02044 VTT, Finland.
Jäntti
Jussi
J
Heilimo
Sara
S
Pihkala
Päivi
P
Paananen
Arja
A
Koskinen
Ari M P
AM
Söderlund
Hans
H
Linder
Markus B
MB
D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D003035:Cobalt; D006639:Histidine; D003300:Copper; D006861:Hydrogen Peroxide; D001205:Ascorbic Acid