title: High level bacterial expression of uteroglobin, a dimeric eukaryotic protein with two interchain disulfide bridges, in its natural quaternary structure.
abstract: Bacterial expression of eukaryotic proteins is a tool of ever-increasing importance in biochemistry and molecular biology. However, the majority of the recombinant eukaryotic proteins that have been expressed in bacteria are produced as fusion proteins and not in their native conformation. In particular, correct formation of quaternary structures by recombinant proteins in bacterial hosts has been reported very rarely. To our knowledge, correct intracellular formation of multimeric structures containing more than one interchain disulfide bridge has not been reported so far. We have constructed three plasmids which are able to direct expression of recombinant rabbit uteroglobin, a homodimeric protein with two interchain disulfide bridges, in Escherichia coli. Among these, the plasmid pLE103-1, in which the expression of recombinant uteroglobin is controlled by a bacteriophage T7 late promoter, is by far the most efficient. With pLE103-1, recombinant uteroglobin production reached about 10% of total bacterial soluble proteins. This protein accumulated in bacterial cells in dimeric form, as it is naturally found in the rabbit uterus. Recombinant uteroglobin was purified to near-homogeneity and its NH2-terminal amino acid sequence was confirmed to be identical to that of its natural counterpart, except for 2 Ala residues the codons for which were added during the plasmid construction. This protein was found to be as active a phospholipase A2 inhibitor as natural uteroglobin on a molar basis. To our knowledge, this is the first report of high level bacterial expression of a full length eukaryotic homodimeric protein with two interchain disulfide bridges in its natural, biologically active form. The plasmid pLE103-1 may be useful to explore structure-function relationships of rabbit uteroglobin. In addition, this plasmid may be useful in obtaining high level bacterial expression of other eukaryotic proteins with quaternary structure, as well as for other general applications requiring efficient bacterial expression of cDNAs.
authors.affiliation: Section on Developmental Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: L
authors.initials: L
authors.lastname: Miele
authors.forename: E
authors.initials: E
authors.lastname: Cordella-Miele
authors.forename: A B
authors.initials: AB
authors.lastname: Mukherjee
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004220:Disulfides / Q000032:analysis; D005260:Female; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014598:Uteroglobin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014599:Uterus / Q000378:metabolism
chemical_list: D004220:Disulfides; D006023:Glycoproteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014598:Uteroglobin