TY - JOUR
T1 - Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host
AU - Chen, Benjamin P C
AU - Hai, Tsonwin
PY - 1994/2/11
Y1 - 1994/2/11
N2 - We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [γ-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.
AB - We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [γ-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.
KW - Histidine tag
KW - T7 expression system
KW - heart muscle kinase
KW - nickel-chelating column
KW - protein phosphorylation
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U2 - 10.1016/0378-1119(94)90525-8
DO - 10.1016/0378-1119(94)90525-8
M3 - Article
C2 - 8112591
AN - SCOPUS:0028267319
SN - 0378-1119
VL - 139
SP - 73
EP - 75
JO - Gene
JF - Gene
IS - 1
ER -