Abstract
Rhomboid proteases are membrane-embedded proteases that cleave peptide bonds of transmembrane proteins. They play a variety of roles in cell signaling events. The rhomboid protease GlpG from Haemophilus influenzae (hiGlpG) is a canonical form of rhomboid protease having six transmembrane segments. In this unit, detailed protocols are presented for optimization of hiGlpG expression using the araBAD promotor system in the pBAD vector. The parameters for optimization include concentration of inducing agent, induction temperature, and time. Optimization of these key factors led to the development of a protocol yielding 1.6 to 2.5 mg/liter protein purified after ion metal affinity chromatography (IMAC). Further purification can include size exclusion chromatography (SEC).
Keywords:
IMAC; SEC; expression study; pBAD; rhomboid protease.
Copyright © 2014 John Wiley & Sons, Inc.
Publication types
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Research Support, Non-U.S. Gov't
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Review
MeSH terms
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Bacterial Proteins* / biosynthesis
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Bacterial Proteins* / chemistry
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Bacterial Proteins* / genetics
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Bacterial Proteins* / isolation & purification
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Endopeptidases* / biosynthesis
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Endopeptidases* / chemistry
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Endopeptidases* / genetics
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Endopeptidases* / isolation & purification
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Gene Expression*
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Genetic Vectors*
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Haemophilus influenzae* / enzymology
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Haemophilus influenzae* / genetics
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Membrane Proteins* / biosynthesis
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Membrane Proteins* / chemistry
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Membrane Proteins* / genetics
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Membrane Proteins* / isolation & purification
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Promoter Regions, Genetic
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Protein Structure, Tertiary
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
Substances
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Bacterial Proteins
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Membrane Proteins
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Recombinant Proteins
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Endopeptidases