Protein Expression & Engineering Flashcards
Elements involved in expression vectors
- Promoters = key regions in the production of recombinant proteins to activate successful gene expression; e.g inducible promoters Tetracycline + derivatives, Arabinose, Galactose, Lactose, Rhamnose
- RNA Polymerase = recognises and binds to promoter region and starts the gene expression process; DNA is signalled to unwind so that RNA Pol can read the bases of the gene of interest on the DNA strand and make an mRNA strand with complementary sequence of bases
- Operator = gateway to the promoter to control levels and timings of protein expression; repressor proteins ie. LacI bind to operator, so RNA Pol is no longer able to bind; if nothing is bound to the operator and promoter, a conformational change occurs allowing RNA Pol to bind and gene expression to start
- Ribosome Binding Site (RBS) = a sequence found in mRNA that is recognised by ribosomes, they then bind and initiate translation of the target protein; it also marks the start of the RNA transcript; in prokaryotes, the RBS is called the Shine-Dalgarno sequence
- Autophagy Related Genes (ATG) = mark the start of the gene of interest; start codon ATG ie. Met
- Terminator = after the stop codon and marks the end of transcription
- Antibiotic Resistant Gene = selects individual E. coli cells that have taken up the plasmid during the transformation process
- Replication origin = drives the production and replication of many of these plasmids; amplifies plasmid to ensure the sequence is correct and that there is sufficient DNA for experiments
Recombinant protein
Example of promoters in lac operon
Control and optimization of expression of the target protein
Bacterial growth phases
Limitations and competition to recombinant protein expression
Autoinduction
Plasmid T7 promoter and regulation of T7 RNA polymerase expression
Phage T7 promoter/polymerase
Examples of E.coli strains
Properties of DNA polymerases used for PCR
Obtaining PCR fragment
Cloning PCR fragment using A overhangs
TA-cloning with topoisomerase
Traditional cloning
Ligation independent cloning of PCR fragment
Gateway technology
Fusion protein
Example of E. coli cloning vector
Features and benefits of an E. coli expression vector
Designing primers with restriction site for PCR
Isolation of tagged protein
Eukaryotic expression system
Why is E. coli system not suitable for many proteins
Types of expression systems
Considerations in choosing a system
Pichia pastoris (yeast) expression system
S. cerevisiae (yeast) expression system
Insect cell system
Mammalian cell system
Types of mammalian cell strains
Transfection into mammalian cells
Summary advantages and disadvantages of each system
Controlling gene expression
- Ensure that protein expression is switched on at specific times
- By adding lactose (IPTG); IPTG binds specifically to the LacI molecule shown, occupying LacI’s binding sites so it can no longer bind to the operator region
- Without binding, the promoter adopts a different conformation so RNA Pol can bind; now, transcription and translation of LacZ, LacY, and LacA genes can take place
- Used extensively in bacterial expression
- Can optimise protein expression by adding an inducer that turns the lac operon on
