title: High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris.
abstract: Hyaluronidase from honey bee was recombinantly expressed as a secreted glycoprotein in Pichia pastoris. The active enzyme was produced in milligram quantities per liter of primary culture. When changing the codons of the original transcript to triplet sequences preferred by P. pastoris, no further increase of protein product could be achieved. After expression of a fusion protein by linking hyaluronidase and human serum albumin together with the recognition sequence for the protease, factorXa, fragmented protein products were obtained in the culture supernatant. Only after replacement of the hinge region with a serine-glycine-rich linker, stable full-length fusion protein could be generated. The protein products were purified by cation exchange chromatography at pH 5.0 and pure enzyme fractions were further characterized in detail. The biochemical properties of the product matched those of crude hyaluronidase within bee venom: the native and the recombinant enzyme exhibited activity over a pH range from 3 to 8 (maximum: 3.8), at temperatures as low as 4 degrees C and up to 90 degrees C (maximum 62 degrees C), and at ionic strength as high as 2 M salt. Recombinant bee hyaluronidase efficiently degrades 6-S-chondroitin sulfate (chondroitin sulfate C) as well as 4-S-chondroitin sulfate (chondroitin sulfate A), the latter to a lesser extent. Only very little hydrolase activity towards chondroitin sulfate B (dermatan sulfate) was detectable.
authors.affiliation: The Extracellular Matrix Research Group, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Reitinger
authors.forename: Thorsten
authors.initials: T
authors.lastname: Boroviak
authors.forename: Gerhard Thomas
authors.initials: GT
authors.lastname: Laschober
authors.forename: Christine
authors.initials: C
authors.lastname: Fehrer
authors.forename: Johannes
authors.initials: J
authors.lastname: Müllegger
authors.forename: Herbert
authors.initials: H
authors.lastname: Lindner
authors.forename: Günter
authors.initials: G
authors.lastname: Lepperdinger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001516:Bees / Q000201:enzymology* / Q000235:genetics; D003062:Codon / Q000235:genetics; D018076:DNA, Complementary; D005822:Genetic Vectors; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012709:Serum Albumin / Q000378:metabolism
chemical_list: D003062:Codon; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012709:Serum Albumin; D006821:Hyaluronoglucosaminidase