Abstract
The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin alpha-sarcin to be used for immunoconjugate production. alpha-Sarcin cDNA was isolated from Aspergillus giganteus strain MDH 18,894 and its expression in Escherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant alpha-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernatant. A variant form of alpha-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.
MeSH terms
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Amino Acid Sequence
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Aspergillus / genetics
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Base Sequence
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Blotting, Western
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Chromatography, High Pressure Liquid
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Cytotoxins / biosynthesis*
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Cytotoxins / isolation & purification
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Endoribonucleases / biosynthesis*
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Endoribonucleases / genetics
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Endoribonucleases / isolation & purification
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Escherichia coli
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Fungal Proteins*
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Genetic Vectors
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Immunoconjugates / isolation & purification
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Molecular Sequence Data
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Protein Synthesis Inhibitors / isolation & purification
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Recombinant Fusion Proteins / biosynthesis
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Staphylococcal Protein A / biosynthesis
Substances
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Cytotoxins
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Fungal Proteins
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Immunoconjugates
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Protein Synthesis Inhibitors
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Recombinant Fusion Proteins
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Recombinant Proteins
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Staphylococcal Protein A
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alpha-sarcin
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Endoribonucleases