RRC ID 33435
Author Muesing M, Carpenter CD, Klein WH, Polisky B.
Title High-level expression in Escherichia coli of calcium-binding domains of an embryonic sea urchin protein.
Journal Gene
Abstract 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.
Volume 31(1-3)
Pages 155-64
Published 1984-11-1
DOI 10.1016/0378-1119(84)90206-3
PII 0378-1119(84)90206-3
PMID 6098520
MeSH Animals Bacteriocin Plasmids Calcium / metabolism Cloning, Molecular DNA, Recombinant Escherichia coli / genetics Escherichia coli / metabolism Gene Expression Regulation Genetic Vectors* Muscle Proteins / biosynthesis Muscle Proteins / genetics* Muscle Proteins / metabolism Repressor Proteins / genetics Sea Urchins / embryology Sea Urchins / genetics* Troponin / genetics Troponin C
IF 2.638
Times Cited 16
Prokaryotes E. coli pMAM17