Improved expression and evaluation of polyethyleneimine precipitation in isolation of recombinant cysteine proteinase inhibitor stefin B.
Synthetic gene coding for human cysteine proteinase inhibitor stefin B was expressed in Escherichia coli by the use of pKP1500 plasmid-containing tac promotor and temperature-sensitive origin of replication, ensuring high plasmid copy number. Several parameters were varied in order to maximize the yield of inhibitory active protein: distance between RBS and initiator codon, temperature of fermentation, and conditions of fermentation. Production of stefin B was markedly improved by setting the RBS to ATG codon distance to 10 nt and with fermentation conditions that increased yield of biomass. The isolation procedure was modified by including precipitation with polyethyleneimine that removed contaminants such as nucleic acids and most bacterial (predominantly acidic) proteins. Precipitation itself produced more than 80% pure recombinant inhibitor, which was purified to homogeneity by a single chromatographic step. Isolated protein had the same inhibitory properties as authentic inhibitor.
Jerala
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Department of Biochemistry, Jozef Stefan Institute, Ljubljana, Slovenia.
Kroon-Zitko
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Turk
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D001483:Base Sequence; D011232:Chemical Precipitation; D003062:Codon; D055313:Cystatin B; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D005285:Fermentation; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011094:Polyethyleneimine; D012333:RNA, Messenger / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D013696:Temperature
C524994:CSTB protein, human; D003062:Codon; D015891:Cystatins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D055313:Cystatin B; D011094:Polyethyleneimine