X ray crystallography Flashcards
what is the fundamental basis of all life?
- chemical interactions
whats the difference between in the old days and now with DNA research?
- in the old days we could only work with abundant proteins, ligands and substrates ( things easy to obtain)
(e. g. hen egg white - lysozyme ) - today - recombinant DNA technologies are used - genes encoding the protein of interest can be cloned and inserted into a plasmid ! ( enhancing protein expression)
- today we only need a very small amount of the material for DNA technology !!
- today we can isolate, purify , exploit PCR etc, we can select and work on exactly what we need.
what are the most commonly used expression system?
-E.coli
- human embryonic kidney cells
( anything that can be cultured in large quantities)
How are cells produced for research?
- these cells are grown in defined media then labelled ( for NMR) or chemically modified ( for XRAY phasing) amino acids can be added into the protein of interest
What are the steps included in getting more cells of interest…?
1- the expression cells are cultured
2- the protein expression is then induced
3- the cells are harvested by centrifugation
4- broken open and the desired material is removed for the next stage which is
PURIFICATION !!!
What does the process of Purification involve?
1 - plasmids can introduce a tage onto the protein product ( either N or C terminus )
2-this can be used to form a tagged or fusion protein - (e.g. histidine Tag or a glutathione S- transferase fusion )
3. a tagged protein ( eg histidine tagged protein ) can be passed over a metal ion chelating column (Ni2+)
4. the protein then sticks to the column - everything else passes through
5. the protein target is then eluted using an imidazole wash ( which can achieve a 95% purity from this single column )
N.B other chromatographic methods can be used to puritfy this further - the product can be cleaved to remove the tag and to leave the protein target ( this can be done if a protease cleavage site has been engineered into the expression system — it isused to cleave Histidinde tag so no fragments are left on the metal ions )
What are the steps in preparing an E.coli Recombinant expression system?
1.the antibiotics resistance gene is in the vector
2- restriction enzyme used to cut at multiple cloning sites on the vector
3. the gene of interest is placed into the vector ( with the antibiotics resistance gene !) ( DNA Ligase used )
4. expression of plasmid
5,. E.coli and plasmid mixed together - under good conditions the E.coli will take up that gene.
6,the E.coli cells are then plated and then further innoculted
7. sometimes you get the gene of interest in the plasmid , or other times the plasmid reseals without it.
N.B. if the expression vector carries a system to allow for colour change - then we see whether the plasmid does what we want - e.g. production of a bluc colour - when B- galactosidase is present OR a white colour - when the enzyme is absent - this allows for selection !
What is IPTG ?
- isopropyl B-D-1 Thiogalactopyranoside )
- used to switch on transcription of the lac operon, which induces the expression of any gene under the control of the lac operator.
IPTG - binds to the lac represser and so switches off repression , rather than switches on transcription.
( it turns off ‘molecules’ which prevent genes turning on !! )
Once the gene of interest has been found , how can more be made?
- cloned from template or cDNA by PCR
mutants can be made again using PCR or DNA can be synthesized ready for use!
Why might we not use E.coli as a system ?
- post translational modifications !
Explain Purification in terms of speed and efficiency?
- recombinant Protein has been overproduced
- cells are broken open by mechanical (e.g. sonicator , french press- large amount of pressure)
- high speed centrifugaation and use of nucleases, filtration can be combined to remove cell debris. -( cell gunk !- reducing viscosity - e.g. E.coli when broken down is like a big viscous soup!!!)
- if secreted the media is concentrated and the protein may be present at a high level of purity !
- first chromatography step - affinity column
- could use a column with antibody - ( a lectin to breakdown the glucose ) - for glycoproteins , transistion metal ions (Ni 2+ )
PURITY IS ESSENTIAL TO AVOID ARTIFACTS !!
What is Chromatography?
- Chromotography:- separation between a mobile and a stationary phase
( the sample in buffer= mobile phase , the stationary phase = solid media, spherical particles packed in column )
the stationary phase allows for differential separation by chemical attraction , based on a specific interaction , charge , hydrophobicity or size.
( molecules with small size/charge can be separated as well !!)
What are four methods of Chromatography - and what are their physical/chemical property and resolution?
- Ion exchange adsorption - charge - high
- size exclusion( gel filtration ) - size/shape - low
- works from high to low- - Hydrophobic interactions -surface hydrophobicity - medium
- Affinity Chromatography - (Bio) Chemical Binding - high
Describe the basic schematic setup of purification ?
( refer to diagram in lecture 1)
What is Affinity Chromatography ?
-method of separating biochemical mixtures based on a highly specific interaction such (e.g.antigen and antibody, enzyme and substrate, or receptor and ligand.)
- one kind of molecule in the mixture is attached to the molecule that is linked on the resin , other molecules washes through
Two common affinity tags :
- Histidine Tag
( agarose beas linked to NTA ( nitrilotriacetic acid))
- Glutathione S. Transferase fusion ( GST tag)
( agarose bead linked to glutathione binds to this tag)
What is Ion Exchange Chromatography ?
the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
the net surface charge of a protein varies according to pH
pH> isoelectric point (PI) = protein +ve ( anion exchanger )
pH< isoelectric point (PI) = protein -ve ( cation exchanger )
this method involves adsorption to the charged solid resin /support followed by elution in a buffer of higher ionic strength.
( concentrated salt solution can be used to wash the protein away from the medium - charge can be used to separate also.)
What is Hydrophobic interaction ( chromatography )?
- it is a harsh method- can cause trouble with many samples ( was used a lot more commonly in the olden days, affinity chromatography much more common now)
- HIC is based on the reversible interaction between a protein and the hydrophobic ligand bound to the chromatography matrix.
- a matrix
- it depends on how hydrophobic your protein is.
a matrix containing hydrophobic groups binds proteins from aqueous solutions to different extents - depending on the protein structure and a range of other factors ( e.g. salt concentration , pH , temperature)
What is size exclusion Chromatography ?
molecules in solution are separated by their size, and in some cases molecular weight.- usually applied to large molecules such as proteins .
- gel filtration or molecular seive chromatography
- used to investigate protein to protein interactions
partition chromatography to separate molecules of different sizes
porous gel mix - in the form of beads - are used!
- smaller molecules enter matrix pores and move more slowly through the column
molecules of intermediate size can enter the stationary phase - travelling quickly through
- with the correct use of a calibrated column then we can elucidate the mass of the protein = hence its Quaternary structure in solution
gel filtration - larger molecules goes straight to bottom
smaller take longest time.
what methods do we do to assess the level of purity ?
- enzyme or binding assay for activity
- SDS PAGE and Mass Spectometry
- Bradford assay or UV/VIS Spectroscopy for concentration.
