Expression of cDNAs encoding wild-type and mutant neuromodulins in Escherichia coli: comparison with the native protein from bovine brain.
Murine cDNA that encodes neuromodulin, a neurospecific calmodulin binding protein, was inserted into the plasmid pKK223-3 for expression in Escherichia coli. After being transformed into E. coli strain SG20252 (lon-), the expression vector directed the synthesis of a protein that was recognized by polyclonal antibodies raised against bovine neuromodulin. The recombinant protein expressed in E. coli was found to be tightly associated with insoluble cell material and was extractable only with guanidine hydrochloride or sodium dodecyl sulfate. Following solubilization with guanidine hydrochloride, the protein was purified to apparent homogeneity by a single CaM-Sepharose affinity column step with a yield of 0.2 mg of protein/L of E. coli culture. The availability of the purified recombinant neuromodulin made it possible to answer several specific questions concerning the structure and function of the protein. Despite the fact that murine neuromodulin is 12 amino acid residues shorter than the bovine protein and the recombinant protein expressed in E. coli may lack any posttranslational modifications, the two proteins displayed similar biochemical properties in almost all respects examined. They both had higher affinity for CaM-Sepharose in the absence of Ca2+ than in its presence; they were both phosphorylated in vitro by protein kinase C in a Ca2+- and phospholipid-dependent manner; neither form of the proteins was autophosphorylated, and the phosphorylated form of the proteins did not bind calmodulin. The recombinant neuromodulin and neuromodulin purified from bovine brain had similar, but not identical, affinities of calmodulin, indicating that the palmitylation of the protein that occurs in animal cells is not crucial for calmodulin interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Pharmacology, University of Washington, Seattle 98195.
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Chapman
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Nicolson
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D019922:GAP-43 Protein; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D004269:DNA, Bacterial; D019922:GAP-43 Protein; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA