Abstract
Complementary DNAs encoding human stefins A (HSA) and B (HSB) were synthesized using Pichia-preferred codons by overlap extension PCR. The full-length genes were ligated downstream of the glyceraldehyde-3-phosphate dehydrogenase promoter in the Pichia expression vector pGAPZαC and successfully expressed in Pichia pastoris strain X-33. Functional recombinant HSA and HSB proteins were purified from culture medium at yields of 121.3 ± 13.5 (n = 3) and 95.4 ± 4.1 (n = 3) mg/L, respectively. Using this expression strategy, we demonstrated that high levels of bioactive recombinant HSA and HSB can be produced by fermentation in P. pastoris.
© 2013 International Union of Biochemistry and Molecular Biology, Inc.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amyloidogenic Proteins / genetics*
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Amyloidogenic Proteins / metabolism
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Base Sequence
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Codon / genetics*
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Cystatin A / genetics*
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Cystatin A / metabolism
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Cystatin B / genetics*
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Cystatin B / metabolism
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DNA, Complementary / genetics*
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Fermentation / genetics
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Genetic Vectors / genetics
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Glyceraldehyde-3-Phosphate Dehydrogenases / genetics
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Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
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Humans
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Molecular Sequence Data
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Pichia / genetics*
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Pichia / metabolism
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Promoter Regions, Genetic / genetics
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
Substances
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Amyloidogenic Proteins
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CSTB protein, human
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Codon
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Cystatin A
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DNA, Complementary
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Recombinant Proteins
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CSTA protein, human
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Cystatin B
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Glyceraldehyde-3-Phosphate Dehydrogenases