Lecture 2 Flashcards
What are the things to consider before purifying a protien
The protien source
The way it’s expressed
The tags you will use on it
The way to cleave the tag
The potential conditions the protein is in to stop it from lysing and what it needs to stay intact
Do we need a second affinity chromatography step to get rid of the tag when it’s cleaved
What are the types of fusion tags
MBP
GST
6xHis
What is mbp
How is elution performed
a maltose binding protien that binds to amylose or maltose
Fist use a amylose resin so protien with mbp tag binds, use wash buffer to get rid of useless proteins, use maltose and buffer to release purified protein
The maltose outcompetes with the amylose
What is GST
How is elution performed
It binds to glutathione (GSH) via redox reaction to form a dimer
Using GSH to out compete the column and wash the tagged protien out or low pH
What is 6xHis
How is elution done
A string of 6 histidines in the protein
The imidazole of the his binds to nickel cobalt and zinc
Elite With imidazole or low ph
What are the most common protien separation methods
IMAC: immobilized metal affinity chromatography
IEC: ion exchange chromatography
SEC: size exclusion chromatography
Or cleave with a protease
What is endogenous and recombinant source of protien
Endogenous is where the protien came straight from the native tissues/cells (like from cow eyes)
Recombinant: the protien is genetically engineered via recombination (being over expressed in an expression system)
Which has a simpler purification : tissue lysates or recombinant expression systems
The expression systems because the lysate requires you to remove other cellular protiens through different methods
The expression system only needs engineered tags to purify
What are the expression systems and their pros and cons
Bacterial: good for making fast expression and cheap, but some eukaryotic protiens might not fold or get the proper PTMs
Yeast: good for SOME eukaryotic PTM (glycosylation) still fast and cheap
Insect cells: used for expressing protiens with complex folding requirements and complex environment for PTM
Mammalian cells: good for protiens that need human like PTMS, more expensive and slower than bacteria and yeast
Which type of tags are ph dependent
GST and 6xHIS
What are the different lysis conditions of protiens
The buffer ph: affects the protien solubility and stability, should match optimal protien conditions
Reducing agents: prevent disulphide bond formation during purification (don’t want broken protien to come back together through oxidizing)
Protease inhibitors: used to prevent degredation of the target protien by endogenous proteases during the lysis and purification steps
What are the requirements of proteins to consider during purification
Metals: some protiens need metal ions (zinc) for stability or enzymatic activity, so they need to be in the buffer during purification
Cofactors: protiens that usually interact with the small cofactors or molecules during their normal function, usually need them for proper folding or activity
PTMS: phosphorylation, glycosylation, or lipidation are required for proper protien folding (epasaiclly in mammal protiens)
What happens if you need to cleave a fusion tag that you’ve added
After the protien is purified it still has a his tag
You do a second affinity chromatography step after cleaving the tag to separate the protien from the tag and the tag protease in solution
This second step can be adding another metal chromatography column, his tag bind to it and out target protein flows through
What is hydrophobic interaction chromatography
You have uv absorbance through trp and tyr residues in the protiens
You have a hydrophobic column
Having high salt concentration makes the protiens more sticky and they bind to the column.
As salt decreases, the stickiness decreases and the less hydrophobic protiens elute faster
What is hydrophobic interaction chromatography
What confributes to the uv absorbance
You have uv absorbance through trp and tyr residues in the protiens
You have a hydrophobic column
Having high salt concentration makes the protiens more sticky and they bind to the column. (High salt, all bind)
As salt decreases, the stickiness decreases and the less hydrophobic protiens elute faster
