A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins.
Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity. Proteins fused in frame with CBP can be purified from crude E. coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992). Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications. To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n. The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E. coli. The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter. A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest. The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E. coli cells at 15-20 mg/1 culture. CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin. Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin. We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase. Hence, the purified fusion proteins can be labeled directly with [gamma-32P]ATP and used to probe protein-protein or protein-nucleic acid interactions.
Zheng
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Stratagene Cloning Systems, La Jolla, CA 92037, USA.
Simcox
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Xu
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Vaillancourt
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D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D006657:Histones; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D013917:Thrombin