title: Stable, high-level expression of a type I antifreeze protein in Escherichia coli.
abstract: The type I antifreeze proteins are simple amphipathic helical proteins found in abundance in polar fish species, where they act to prevent freezing of internal fluids by a mechanism of noncolligative freezing point depression. Large-scale production of these proteins for research and biotechnological purposes has been hampered by their apparent instability when expressed in heterologous host systems. This has necessitated their production as fusion proteins, in polymeric form, or as proproteins for secretion, with the concomitant necessity for postpurification processing to generate the mature form of the protein. We have successfully expressed a recombinant variant of type I antifreeze protein (rAFP) in Escherichia coli using the inducible T7 polymerase transcription expression system. The rAFP contains five copies of the 11 amino acid ice-binding repeat motif found in all type I antifreeze proteins. The protein accumulates to high levels intracellularly in the form of inclusion bodies, with no apparent degradation by the cellular proteolytic machinery. We have devised a simple and rapid purification protocol for this recombinant type I antifreeze protein which does not require cellular fractionation, purification of the inclusion bodies, or chromatographic steps. This protocol may be of general use for this class of protein. The protein displays all three activities common to these proteins: recrystallization inhibition, noncolligative freezing point depression, and modification of the morphology of single ice crystals in solution.
authors.affiliation: CSIRO Plant Industry and Quality Wheat CRC Ltd, Canberra, ACT, 2601, Australia.
authors.forename: R G
authors.initials: RG
authors.lastname: Solomon
authors.forename: R
authors.initials: R
authors.lastname: Appels
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005399:Fishes / Q000235:genetics; D005432:Flounder / Q000235:genetics; D005615:Freezing; D015870:Gene Expression; D005813:Genes, Synthetic; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007053:Ice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020080:Tandem Repeat Sequences
chemical_list: D021301:Antifreeze Proteins; D017931:DNA Primers; D006023:Glycoproteins; D007053:Ice; D011994:Recombinant Proteins