Gene to protein
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E. coli, Pichia pastoris
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- Clone the gene of interest into chosen expression vector.
- Small-scale expression screening. Large-scale (≥1 L) expression culture. Primary protein purification.
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Molecular Cloning and Mutagenesis
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Cloning
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- Clone the gene of interest into chosen expression vector. DNA sequence verification.
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Mutagenesis
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- Single or multiple nucleotide base change e.g. amino acid substitution, Insert / delete restriction site, stop codons and small tag. DNA sequence verification.
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Plasmid production
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- Production and purification of plasmid DNA from mini- to mega-prep scale.
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Small-scale Expression Screening
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E. coli
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- Expression screen under multiple conditions. Each construct tested in two different expression strains, two temperatures and two media and two inducer concentrations.
- Analyze protein expression and affinity binding by SDS-PAGE.
- Resin-binding assay by affinity chromatography. Determine optimal expression conditions.
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Pichia pastoris
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- Expression screen of 10 yeast clones per construct. Analyze expression and affinity binding by SDS-PAGE.
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Large-scale Protein Expression and Purification
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E. coli or Pichia pastoris
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- Expression at ≥ 1 L in shake flasks. Cells and/or supernatant collected at optimal time of harvest. SDS-PAGE analysis.
- If required, we can produce Isotope labelled protein for NMR or mass spectrometry applications.
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Primary purification
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- Sample extraction followed by affinity chromatography (e.g. IMAC, GST, MBP, Strep(ll)). SDS-PAGE analysis.
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Secondary purification
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- Secondary purification to improve protein purity by techniques such as affinity, ion exchange, hydrophobic interaction or size exclusion chromatography. SDS-PAGE analysis.
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Removal of Fusion tag
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- Screen for optimal protease cleavage condition to remove fusion tag from protein. Chromatography to recover untagged protein. SDS-PAGE analysis.
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