Structural Biology - Protein Expression Flashcards
Explain the genetic elements involved in the production of a plasmid encoded protein
Promotor: DNA sequence bound by RNA polymerase. Drives production of RNA transcript
Operator (downstream of promotor): Control region that binds specific molecules. Leads to a conformational change in DNA that prevents/allows access of RNA polymerase to the promotor
Ribosome binding site: section of DNA that encodes first part of RNA transcript
ATG start site: start of gene of interest
Gene of interest
Stop codon: where translation should stop
Terminator region: where transcription should stop
What are the extra genetic elements in the expression of a plasmid encoded protein other than the part that produces the RNA transcript?
Antibiotic resistance genes: selection of bacterial colonies that contain the plasmid of interest
Replication origin: allows amplification of DNA plasmid within cells
Explain the Lac Operon
Expression of proteins LacI, LacZ, LacY, LacA
LacI is under control of a constitutive promotor
LacI is a repressor (RNA polymerase can’t bind) that controls expression of other Lac proteins by binding to the operator
Lactose IPTG (analogue of lactose) binds to LacI and acts as an inhibitor by inducing a conformational change so no longer binds to operator
Then get transcription and translation of 3 lac proteins
Proteins like LacI are sensitive to sugars (arabinose, galactose) or tetracycline which can be used to drive the production of a protein
Describe the stages of cell growth (OD vs time graph)
Lag Phase (hour 1): inoculate culture containing low number of cells, low growth rate
Exponential phase: rapid growth, inducer molecule added at OD = 0.6
lactose added to induce expression of a target gene
Stationary phase: cells still growing but also dying rapidly, no net growth, resources used up
Death phase
How can expression of cells be optimised?
Vary concentration of inducer molecule
Vary temperature of growth (18 vs 37 degrees)
What is autoinduction?
Does not rely on the addition of an inducer molecule
Medium contains glucose and lactose
Glucose (or a specific carbon source) inhibits expression of target gene but as it is consumed, lactose becomes primary carbon source
Induction of target protein
What is cytotoxicity?
Expression of recombinant proteins can be toxic because a small amount of protein is expressed before the culture is ready
Avoid toxicity by separating cloning of gene from gene expression
Explain how T7 polymerase expression is regulated
T7 polymerase is provided by a separate plasmid (expression plasmid).
It contains the gene of T7 polymerase under control of a different promotor, like Lac
Induce Lac promotor by adding IPTG, leading to expression of polymerase
T7 polymerase binds to promotor pT7 to produce target molecule
List examples of E. coli expression strains
BL21: deficient in proteases, better at producing non-native proteins
BL21/STAR: Deficient in RNase, improves stability of mRNA transcripts
BL21(DE3): Reduces expression of recombinant genes, lowers amount of target protein produced before you want it to be
BL21-AI: Different promotor, fine control of protein produced
BL21 Codon: expresses codons that are usually not expressed in E. coli
BL21 trxB: helps express a disulphide bond in cytoplasm
Explain the properties of DNA polymerase in PCR?
Taq polymerase lacks 3’-5’ exonuclease activity (proofreading) = high error rate, must sequence product at end to check for errors
Taq adds a single A base overhang at 3’ end, useful for cloning
Relative processivity is high (amount of nucleotides added before polymerase dissociates
Polymerases with proofreading activity are slower
How does A-overhang cloning work?
PCR product is blunt ended
Incubate PCR product with Taq polymerase at 75 degrees
Additional A base attaches at 3’ end
A overhang is used to clone PCR product into a plasmid with a T overhang due to complementarity
Non-directional insertion
How does Topo TA-Cloning work?
Topoisomerase I cuts DNA at specific CCCTT sequence
Forms a covalent bond with phosphate group of last T base
Stable interaction so no reformation of the bond
Topo TA cloning vector is fixed with Taq amplified PCR product that has an additional A base
A bases attack bond between T base and topoisomerase in vector
Topoisomerase is released
Non-directional
How does traditional cloning work?
Plasmid contains restriction sites in the MCS
Design primers with extensions encoding the restriction sites
ex. Nco1 site at 3’ and HindIII site at 5’
Double digest both plasmid and PCR product with restriction enzymes
Produces sticky ends so plasmid doesn’t undergo self ligation
Directional insertion
How does ligation independent cloning work?
No restriction digestion
Inserts are amplified by PCR
Design primers that incorporate onto end of gene of interest. Must share 15bp of identical sequence with each end of vector
Linearise vector by PCR
Use T4 DNA polymerase in presence of dGTP (removes bases until reaches first G base to produce overhang)
Repeated with gene of interest and dCTP to create overhangs that overlap
How does gateway cloning work?
Rapid insertion
Using DNA recombination in vitro and a recombinase
Can use different expression systems - can clone gene into different vectors depending on the function (ex. purification, antibody production)
Explain the expression of a protein of interest as a fusion protein
Protein of interest is fused to a tag
Incorporate protease recognition site to be able to cleave off the tag
What are some commonly used tags?
His tag, Maltose binding protein, Glutathione S-transferse, GFP
Maltose binding protein
Purification
Enhanced solubility
Very big
Amylose resin/maltose matrix
What are some features and their functions in an E. coli expression vector?
T7 promotor: T7 polymerase binds to
RBS: Ribosome binds
ATG: translation initiation
6x His Tag: detection of recombinant protein
Epitope tag: Detection of fusion protein
Enterokinase cleavage site: Site for removal of fusion tag
MCS: insertion of gene of interest
His Tag
Used for detection and purification
short tag so low influence on fold of overall protein
divalent metal (Ni2+)/imidazole or low pH matrix
GST
Purification
Enhanced solubility
Quite big
Glutathione agarose matrix
GST dimerisation might affect fusion properties
GFP
Used for detection
How do you design a forward primer (5’ to 3’)?
At start of DNA sequence
Take start of 5’ to 3’ strand
Tag on additional DNA that encodes a restriction site (BamHI) at 5’ end
End on a C or G base (3 H bonds increase annealing)
How do you design a reverse primer?
At end of DNA sequence
Consider 5’ to 3’ strand
Use complement of strand to create 3’ to 5’ strand
Tag on to the 5’ end the sequence encoding restriction site for other enzyme (HindII)
Add additional sequence at 5’ on both reverse and forward primer to allow enzyme to bind and cleave (GCG)
How do you isolate a His tag using metal affinity chromatography?
Bind protein of interest to Ni2+ NTA column
Washing steps to remove any non specific proteins
Elute with imidazole
Proteolytic degradation to remove the His-tag
Load material on a gel
Get 2 bands (one his tag, one non his tag protein)
Use non his tag protein to grow crystals
