structural biology Flashcards
cellular environment vs purified protein
purified protein gives more controlled environment, easier tot assay without background noise, this doesn’t fully reflect cellular environment, consequences on assays and structural approaches
considerations when expressing a protein
how do we get sufficient quantities to study it, how much do we need, is it toxic to the cell, what does it do/regulate, is it in membrane or soluble, what post translational modifications may be required
bacterial cell expression
advantages - cheap, easy to grow, easy to manipulate, fast doubling rate, genome is often well characterised and can be scaled up, disadvantages - hard to express non material proteins
bacterial cell expression vectors
for bacterial cell expression important that the plasmid has the following - bacterial origin of replication, antibiotic resistance, transcriptional promoter (controls binding of RNA polymerase), multiple cloning sites, ribosome binding site (so ribosome can bind and transcription initiated), translation terminator
bacterial lines
DH5 alpha - mutated in recA1 (inactivate homologous recombination), endA1 (inactivates endonuclease to prevent plasmid degradation and LacZM15 (blue white screening), BL21 - standard strain deficient in certain proteases snd can use the standard tac promoter system, BL21-(DE3) - uses T7 polymerase system giving a tight control of expression, BL21-Rosetta - also contains a pRARE plasmid to overcome problems with rare codons
monitoring growth
in most instances the growth phase (exponential phase) used for target gene expression to maximise the amount of protein produced, in some cases stationary phase can be used `
yeast cell expression
advantages - cheap easy to grow, easy to manipulate, genome is often all characterised and easy to scale up, eukaryotic cell, disadvantages - doesn’t to work for all eukaryotic proteins, can be hard to get protein out
yeast cell expression vectors
for yeast cell expression important that the plasmid has the following - URA3/ampicillin antibiotic selection, multiple cloning site, under the Gal1 promoter, CYC1 TT terminator site, pUC1 ori origin of replication for bacteria
HEK cell expression
advantages - post translational modifications, good human cell environment mimic, disadvantages - stable cell lines take a long time to generate and transient transfection costly and can be inefficient, the cells can be easily contaminated
HEK cell expression vector
for HEK cell expression important that the plasmid has the following - multiple cloning site, antibiotic resistance gene, compatible promoter, optional tag (SV40) and bacterial origin of replication (f1 ori)
insect cell expression
advantages - post translational modifications, can be scaled and the virus stocks can be down, disadvantages - can be costly and times consuming, can easily get infection of the growth
insect cell expression - process
transformation of competent E coli with gene of interest, selection and expansion of positive clones, isolation of plasmid/expression vector, co-transfection of insect cell lines, production of high titer recombinant virus stock, infection of insect cells with high titer recombinant virus stock; isolation of proteins of interest
cell free expression
advantages - can do membrane proteins and incorporate non natural amino acids and other labels, avoids toxicity, no proteases, disadvantages - very costly, not trivial to set up, can lack some post translational modifications - done as batch, continuous change or continuous flow
native sources
advantages - natural environment and associated proteins, no artefacts of expression, for membrane proteins have natural lipid associated and post translational modifications are present, disadvantages - many proteins are in low abundance, no natural tags, often non optimal tissue for sample prep
mechanical extraction
uses force to break open the cell and includes mortar and pestle, blender, bead beating, ultra sonication and homogenisation
mechanical - bead beating
glass or ceramic beads used to crack open cell, common used for yeast
mechanical - homogenisation
cells are lysed by forcing them through narrow space, uses shear force similar to bead beating
mechanical - ultra sonication
induce vibration within titanium probe immersed in the solution, forms tiny bubbles and explode producing local shockwave (cavitation)
non mechanical extraction - freeze/thawing
series of freeze thaw cycles ice crystals form which expand upon thawing and cause cells to rupture, why its hard to freeze fruit
non mechanical extraction - microwave/thermolysis
temperature used to disrupt bonds within cell wall, can also denature the protein you’re interested in, thermophilic proteins perfect for this method
non mechanical extraction - osmotic shock
use osmosis to increase size of cell till bursts, only used on animal cells and protozoa - don’t have cell wall
non mechanical extraction - chemical methods
ether, benzene, surfactants etc used to solubilise cell membrane causing lysis, for bacteria EDTA used with chelate the Ca2+ and in turn destabilises the lipopolysaccharides leaving holes in the cell wall
non mechanical extraction - enzymatic methods
use digestive enzymes to decompose the microbial cell wall, enzymes will depend on cell type as each have different walls/membranes, example is zymolase used for yeast to degrade tough cell wall
tag purification - His tag
most common purification tag is His tag which is simply 6 to 10 His residues at the N or C terminus
tag purification - peptide/epitope tag
typically 8 to 12 amino acids in length and correspond to a specific immunoaffinity tag, one of most common is FLAG tag (typically Asp-Tyr-Lys-Asp-Asp-Asp-Lys)
tag purification - folded protein tag
include MBP and GST, columns can be used that are highly specific for these tags, one advantage is they can enhance target solubility
tag purification - precipitation/aggregation tags
these can be an effective an inexpensive way of purifying a protein and can have high expression yields with aggregation tags, doesn’t not need any sort of purification column but the tags are often large and can require reflowing protocols (RTX tag undergoes reversible precipitation in response to Ca2+
tag purification - detection tags
often used for membrane proteins and/or proteins where cellular location is important, commonly a GRP or YFP tag is used which means you can use fluorescence to track the location, presence of the tag/fluorescence is good measure if amount of protein expressed
tag purification - solubility/folding tags
often used to increase solubility of the target as the tags are highly soluble, can be beneficial in refolding of target in some cases, their size can be problematic and decrease overall yield, SUMO (small ubiquitin related modifier) shown to enhance expression and folding of recombinant proteins in prokaryotic and eukaryotic hosts, specific SUMO protease means it can be easily cleaved
combining tags
different tags have different properties and therefore more than one tag might be useful, important consideration is ability to remove the tag as it could interfere with assays and cause false positives or negatives
size exclusion chromatography
separate proteins primarily based on size and shape, purification column contains a resin of beads which contains cavities, the smaller proteins reside longer in the matrix, larger one can pass through quicker (smaller ones get trapped in the cavities for a bit) and elute first
ion exchange chromatography
separates ions and polar molecules based on their affinity to the ion exchanger, proteins will bind to the oppositely charged insoluble stationary phase (resin beads) whilst passing through the column
centrifugations and glycerol gradients
can separate based on size shape and composition, old method but still effective for some protein systems especially larger protein complexes such as ribosomes ATP synthase etc
hydrophobic interaction chromatography
hydrophobic surfaces can attract each other, hydrophobic column can be used to separate proteins based on their hydrophobic nature, by changing buffer composition, in particular salt conc your an change surface characteristics and therefore tune when a protein stays attached to the column
lipid influence of structure and function
membrane not just there to support the protein but can also be important for stability and function, big problem when conducting drug screens snd doing structural biology as the proteins may not be representative of how they are in the cell
how do we study membrane proteins
many options for stabilising membrane proteins - detergents, nanodiscs, and amphipods , many of the extraction strategies involves detergents , these can remove the lipids and destabilise the protein, use of proteoliposomes and nanodiscs can restore some of these properties, the proteoliposome in particular is a good platform for maintaining bilayer
detergents
advantages - well established, larger number of different types so broad application, very effective at extracting membrane proteins and used in range of biophysical techniques, disadvantages - poorly reflect the bilayer of the membrane, viscous properties can slow down/inhibit movement of TM domain and perturb structure, expensive, do not maintain closely associated lipid environment and detergent micelles create problems in downstream biophysical analysis
amphipols
octyl groups form multiple hydrophobic interactions with TM domains, the carboxylic groups give the hydrophilicity/solubility, advantages - extensive hydrophobic interactions result in low Koff and small Kd, once added are not required in other buffers, no background micelles and a large library of polymers for different applications, disadvantages - nature of the polymer makes them sensitive to Ca2+ and Mg 2+ ions, viscous nature can slow down/ inhibit movement of the TM domain and do not maintain the closely associated lipid environment
