High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein.
A plasmid expression vector is described having features that facilitate high-level expression of eukaryotic DNA in Escherichia coli. The vector, designated pMAM17, carries the ColE1 rop gene under the control of the thermally inducible lambda PL promoter. The rop gene product is a negative regulator of ColE1 DNA replication, and its high-level expression is lethal to cells. However, cells harboring a plasmid with an insert in the rop gene grow normally under these conditions. pMAM17 has been used to investigate the properties of a family of proteins expressed in the dorsal ectoderm of sea urchin embryos. The coding sequences of these proteins (termed Spec proteins) have homology to the troponin C superfamily. Large amounts of the Rop-Spec fusion protein were produced at 42 degrees C in E. coli. Unfractionated E. coli extracts containing the fusion protein could be used to produce antibodies that were highly specific for Spec proteins present in crude extracts of sea urchin embryos. Analysis of the Rop-Spec fusion protein on SDS-polyacrylamide gels in the presence and absence of EGTA indicated that the fusion protein bound calcium ions in a manner characteristic of proteins of the troponin C superfamily. This behavior provides biochemical evidence that the Spec proteins are functionally homologous to other members of this superfamily.
D000818:Animals; D003086:Bacteriocin Plasmids; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012617:Sea Urchins / Q000196:embryology / Q000235:genetics*; D014336:Troponin / Q000235:genetics; D019209:Troponin C
D004274:DNA, Recombinant; D009124:Muscle Proteins; D012097:Repressor Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium