High-yield recombinant expression of the extremophile enzyme, bee hyaluronidase in Pichia pastoris.
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.
Reitinger
Stephan
S
The Extracellular Matrix Research Group, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria.
Boroviak
Thorsten
T
Laschober
Gerhard Thomas
GT
Fehrer
Christine
C
Müllegger
Johannes
J
Lindner
Herbert
H
Lepperdinger
Günter
G
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
D003062:Codon; D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012709:Serum Albumin; D006821:Hyaluronoglucosaminidase