title: A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.
abstract: We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.
authors.affiliation: Genetics Institute, Cambridge, MA 02140.
authors.forename: E R
authors.initials: ER
authors.lastname: LaVallie
authors.forename: E A
authors.initials: EA
authors.lastname: DiBlasio
authors.forename: S
authors.initials: S
authors.lastname: Kovacic
authors.forename: K L
authors.initials: KL
authors.lastname: Grant
authors.forename: P F
authors.initials: PF
authors.lastname: Schendel
authors.forename: J M
authors.initials: JM
authors.lastname: McCoy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D016207:Cytokines / Q000235:genetics; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D006133:Growth Substances / Q000235:genetics; D006358:Hot Temperature; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics*
chemical_list: D016207:Cytokines; D004274:DNA, Recombinant; D006133:Growth Substances; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins