Protein Expression & Engineering

Question 1

Elements involved in expression vectors

Answer 1

• 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

Question 2

Recombinant protein

Question 3

Example of promoters in lac operon

Question 4

Control and optimization of expression of the target protein

Question 5

Bacterial growth phases

Question 6

Limitations and competition to recombinant protein expression

Question 7

Autoinduction

Question 8

Plasmid T7 promoter and regulation of T7 RNA polymerase expression

Question 9

Phage T7 promoter/polymerase

Question 10

Examples of E.coli strains

Question 11

Properties of DNA polymerases used for PCR

Question 12

Obtaining PCR fragment

Question 13

Cloning PCR fragment using A overhangs

Question 14

TA-cloning with topoisomerase

Question 15

Traditional cloning

Question 16

Ligation independent cloning of PCR fragment

Question 17

Gateway technology

Question 18

Fusion protein

Question 19

Example of E. coli cloning vector

Question 20

Features and benefits of an E. coli expression vector

Question 21

Designing primers with restriction site for PCR

Question 22

Isolation of tagged protein

Question 23

Eukaryotic expression system

Question 24

Why is E. coli system not suitable for many proteins

Question 25

Types of expression systems

Question 26

Considerations in choosing a system

Question 27

Pichia pastoris (yeast) expression system

Question 28

S. cerevisiae (yeast) expression system

Question 29

Insect cell system

Question 30

Mammalian cell system

Question 31

Types of mammalian cell strains

Question 32

Transfection into mammalian cells

Question 33

Summary advantages and disadvantages of each system

Question 34

Controlling gene expression

Answer 34

• 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