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Picture of purification table with explanations

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Picture of purification table with explanations

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- FPLC
- Vivaflow
- Centrifugation
- Check for virus DNA (viruses)

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- To get an overview of all of the purification step and to know at which step to start optimizing

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- Bacteria
- Biomass
- Spores
- Viruses
- Fungus
- Parasites
- Proteins that are not of interest
- Salts

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- Phosphorylation ->  addition of phosphate group on serine, threonine, and tyrosine. Has influence on cellular processes (cell cycle, growth, apoptosis). Detection via Western blot.

- Glycosylation -> critical to folding, conformation, stability. Addition of a variety of sugars (monosaccharides to polysaccharides). Detection via glycoprotein staining or mass spectrometry.

- Ubiquitination -> addition of ubiquitin to one of the amide groups of lysine via the C-terminal glycine in ubiquitin resulting in polymerization. Polymer is recognized by 26S proteasome which catalyzes the degradation and recycle of ubiquitin. Detection via Western blot.

- S-nitrosylation -> addition of NO, to free cysteine groups in the protein forming S-nitriols, crucial for protein stability and regulation of gene expression. Detection via SDS-PAGE paired with Western blot or Mass Spectrometry.

- Methylation -> transfer of methyl group to amine or carboxylic groups in amino acid. Well known for epigenetic regulation in the cell, related to histone formation. Multiple methyl groups can be conjugated to the same amino acid residue.

- Lipidation -> targeting membrane proteins (cell membrane, vesicle proteins, proteins in membranous organelles). Different modifications influence hydrophobicity increasing membrane affinity. Detection via specific membrane probes.

- N-acetylation -> different types of acetylation, different acetylases have different effects e.g. histone deacetylase decreases acetylation levels and increases chromosomal density. Determination via immunoprecipitation and/or Mass Spectrometry.

- Proteolysis -> done by various proteases e. g. Serine and Cysteine proteases. Process is thermodynamically favorable. Basic mechanism is cleavage of peptide bonds.

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- Signal peptide cleavage
- Proteolysis
- Phosphorylation
- Glycosylation
- Ubiquitination
- Rare or low usage codons -> some organisms use some codons less or can't use them, causing misfold
- Inclusion bodies -> reduce overexpression (use different promotors, reduce expression temperature), add protein folders to the expression (chaperones), consider different host, isolate and renaturate

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- Limit proteolytic activity by protease inhibitor
-  Keep protein on ice
- Limited by removal of cells
- pH 4.5 - 6 -> keep outside of this range to limit activity
- Proteolytic deficient strains