Protein Purification Flashcards
what are the different sources of proteins?
Recombinant Protein Produced in Bacteria
Recombinant Protein Produced in Other Organisms
Endogenous protein from tissue
how are recombinant proteins produced?
gene of interest is cloned into an expression plasmid via a vector
- plasmid is transferred into host cells
- high levels of proteins can be produced in host cells
- protein can be purified for functional studies
what is used as an inducer for T7 promoter?
IPTG
what can be added to a plasmid that can make purification easier?
a tag - for example a His tag
bacterial expression in BL21:
there is a his-tag in this expression vector
what are the advantages of protein expression in E.coli?
- fast growth rate - can generate lots of protein-expressing bacteria very quickly
- can transform bacteria with plasmid DNA rapidly
- relatively cheap
what are the disadvantages of protein expression in E.coli?
- proteins may not fold correctly
- high concentration of protein can be insoluble - inclusion bodies
- lack some post-translational modifications - phosphorylation
what is the general protocol for bacterial protein expression? 6 steps
- transform BL21 E.coli with expression plasmid
- pick a colony to grow from - one that has been selected for using the antibiotic resistance gene
- then start expression with the IPTG
- centrifugation to pellet cells
- lysis of bacterial cells
- protein purification
what is the first step in protein purification?
- lyse bacterial cells without degrading or denaturing your protein of interest
- then centrifuge the cells - the supernatant contains soluble cellular material, including proteins
how is protein degradation prevented?
freeze thawing - prevents the proteases
what chemical is used to lyse the cell?
triton X - non-ionic detergent
what other technique can be used to break cells?
Sonication - this is when the cells are broken using high frequency sound waves
what is the second step in protein purification?
properties of the protein are exploited so that it can be purified
give examples of what properties can be exploited?
- size
- charge
- affinity tag - his-tag?
- hydrophobicity
what needs to be taken into consideration when thinking about a purification technique?
- the recovery of the protein
- purity of the protein
- functionality
- cost of method
how can proteins be tracked throughout the purification process?
- western blotting with immunoblotting
- can also use an assay to measure biological activity - this is used for enzymes in particular
SDS-PAGE and Western blotting separates proteins based on what?
their size
SDS stands for: sodium dodecyl sulphate polyacrylamide gel electrophoresis
need to denature the proteins prior to method - what is used to do this?
SDS
what is the first step?
protein samples are loaded into wells of gel and electric current is applied
- -vely charged proteins migrate to the positive electrode
Proteins move through the mesh of? What is it dependent on?
polyacrylamide - the rate of this depends on size - larger proteins nearer the top and smaller proteins at the bottom
what can be added to SDS-PAGE so that the proteins can be visualised?
coomassie blue
what is needed so that you can determine the molecular weight of the proteins?
reference proteins are used - they have a known molecular mass so unknown masses can be compared against them
what is used in immunoblotting?
- antibodies specific for the target protein
- secondary antibody with a tag for detection is then bound to the primary antibody - tag is commonly fluorescence
- bands are then visualised
what factors can affect protein migration?
Large Post Translational modifications
Proteins are not fully denatures, they migrate as complexes
Inefficient reduction of disulphide bonds
High content of basic amino acids can affect migration