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MSF1- Em > Expression and Engineering > Flashcards

Expression and Engineering Flashcards

1
Q

What is a plasmid T7 promoter?

A

Phage promoter that can be incorporated into the E.coli genome.

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2
Q

What does the T7 promoter in E.coli expression systems do?

A

Allows specific control of gene expression- preventing overproduction of the target protein.

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3
Q

What is the advantage of removing a protease gene from an expression system?

A

Prevents degradation of the target protein immediately after production.

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4
Q

What is the advantage of removing an RNase gene from an expression strain?

A

Increases stability of the mRNA transcript, increasing the protein expression yield.

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5
Q

Why would basal expression levels be reduced in an expression strain?

A

Allows more energy to be available for expression of the target gene.

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6
Q

What feature of E.coli promoters is especially useful for expression?

A

Most E.coli promoters are inducible and can be switched on/off by the addition of an inducer.

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7
Q

What is the problem with disulphide bonds when using an E.coli expression system?

A

S-S bonds are usually only found on the extracellular side of the membrane in E.coli. Strains need to be engineered to allow cytosolic S-S formation, to allow the target protein to remain inside the cell.

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8
Q

How can expression be optimised?

A

By varying inducer concentration and by lowering the temperature of growth from 37 to 18 degrees. This reduces production of genes involved in metabolism and growth- more energy available for target gene expression.

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9
Q

Where are purification tags inserted during expression of a target protein?

A

Either side of the protein of interest.

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10
Q

What is a his tag eluted with?

A

Imidazole
Divalent metal ions
Low pH

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11
Q

What are MBP and GST used for?

A

Purification and increasing the solubility of the target protein.

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12
Q

Why must MBP and GST be removed by a protease before NMR or XC?

A

They are large enough to be detected

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13
Q

What is MBP eluted with?

A

Maltose

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14
Q

What is GST eluted with?

A

Glutathione

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15
Q

What is a GFP tag used for?

A

Detection of the cellular location a protein in vivo.

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16
Q

Name two methods of ligation independent cloning.

A

In fusion and LIC using pMCSG vectors

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17
Q

What is in fusion cloning?

A

Ligation independent. PCR insert shares 15bp of identical sequence with each end of the linearised vector. In fusion enzyme makes ss regions at the ends of the vector and insert- fused together due to 15bp homology.

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18
Q

What does gateway technology involve?

A

Rapid insertion of the gene of interest into vectors via DNA recombination in vitro- using a recombinase.m

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19
Q

What is the LIC procedure?

A

Vector cleaved after TEV protease recognition site. T4 pol + dGTP - exonuclease activity removes bases until the first G, overhang on vector. Gene amplified using primers that are complementary to the overhang. T4 pol + dCTP - exonuclease activity removes bases until the first C, overhang on gene. Anneal vector and gene together- TEV protease site in frame with gene.

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20
Q

What are the advantages of bacterial expression systems?

A

Quick, efficient, cheap. Easy to manipulate.

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21
Q

What are some of the limitations of bacterial expression systems?

A

No PTMs, target protein may be sensitive to host protease, different codon bias, may get incorrect localisation and folding of the target protein.

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22
Q

What eukaryotic expression systems are available?

A

Yeast, insect and mammalian.

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23
Q

What are the advantages of using Pichia pastoris (yeast)?

A

Grows to high density, no homologous recombination (efficient production of target protein), uses a linearised plasmid that can integrate into host genome. Alpha factor secretion signal allows the protein to be targeted to the ER.

24
Q

What is the benefit of using the alcohol oxidase promoter in Pichia pastoris?

A

This is a strong promoter- increased expression yield if the target protein gene is under the control of this promoter.

25
Q

What can be used to test expression levels when using yeast systems?

A

Antibiotic resistance or rtPCR

26
Q

What are the disadvantages of using Saccharomyces cerevisae?

A

Has homologous recombination and incorrectly folded proteins are incorrectly trafficked.

27
Q

How are yeast cells burst?

A

Using a French Press - cell suspension pushed through nozzle at high speed and pressure. Cells hit a metallic target to burst any remaining intact cells. System must be kept at low temp. to prevent protein denaturing.

28
Q

What are some of the mammalian cell lines that can be used as expression systems?

A

HeLa cells, CH0 and 3T3 cells

29
Q

What are the advantages of using a mammalian expression system?

A

Uses a shuttle vector with an extensive MCS. Are more variable- e.g. can have inducible or constitutive promoters

30
Q

What are the disadvantages of using a mammalian cell expression system?

A

Expensive as specialist equipment is required. Not suitable for large scale productions.

31
Q

How can the plasmid DNA be transfected into a mammalian cell?

A

By liposome-mediated uptake, electroporation or calcium phosphate precipitate.

32
Q

What is the liposome-mediated method of transfection?

A

DNA-lipid complex fuses with the cell membrane and is endocytosed. Complex is broken down within the cell, releasing the DNA.

33
Q

How does electroporation allow transfection?

A

Electrical pulse forms pores in the membrane and there is an imbalance in membrane potential. This means DNA moves towards the positive charge inside the cell. Following the pulse, the pores are repaired and the cell contains the DNA.

34
Q

What is the calcium phosphate precipitate method of transfection?

A

Phosphate buffered DNA added to calcium chloride solution. Calcium phosphate precipitate forms and DNA binds to the precipitate surface. Precipitate added to monolayer of cells- some take in the precipitate bound target DNA.

35
Q

What is the difference between transient and stable transfection?

A

Transient- short lived, expression after 48 hours and then cells undergo apotosis.
Stable- long term expression, can use antibiotic resistance as a test for expression.

36
Q

Give an advantage and disadvantage of using cell free expression systems.

A

Advantage- can express proteins that cannot be expressed in other systems.
Disadvantage- expensive, not suitable for large scale productions.

37
Q

What are some of the uses of protein engineering?

A

Identification of protein regions critical to function. Creation of novel proteins with a desired function. Improve catalytic function. Alter substrate specificity. Improve protein stability for structural determination.

38
Q

When is the synthetic gene approach used?

A

Used when a lot of DNA changes need to be made.

39
Q

What is the plasmid based approach for directed mutagenesis?

A

Design primers complementary to regions flanking the mutation site. Daughter strand then contains the mutation.

40
Q

When is the plasmid based approach used?

A

To introduce a single point mutation.

41
Q

Describe the overlap extension method.

A

Design 4 oligos complementary to the dsDNA template. PCR 1 with oligos 1+4, PCR 2 with oligos 2+3. PCR products share an overlapping region, containing the mutation region. PCR 3 with oligos 1+2 to give a PCR fragment with the mutation.

42
Q

When is alanine scanning mutagenesis used?

A

To investigate the roles of individual residues within a protein- can then produce a more stable construct for further analysis.

43
Q

What are residues mutated to during alanine scanning mutagenesis?

A

All mutated to Ala and Ala is mutated to Leu- a mutant is produced for every residue in the protein.

44
Q

What is necessary after alanine scanning mutagenesis?

A

Functional and thermostability assays to assess the effect of mutating a particular residue. Mutations can then be combined to produce a more stable construct, whilst maintaining the proteins function.

45
Q

When is iterative selection used?

A

To find more stable constructs.

46
Q

Describe iterative saturation.

A

Every mutation combination is produced. Single point mutations in WT - most stable are identified. Further mutate the most stable ones to include another point mutation, select the most stable. Iterate until the most stable construct is found.

47
Q

What are the random mutagenesis methods?

A

Chemical mutagenesis, PCR approach and plasmids in E.coli cells.

48
Q

Describe chemical mutagenesis.

A

Plasmids are treated with chemicals that cause DNA damage, e.g. Sodium bisulfite. Amplification by PCR.

49
Q

What is the PCR approach?

A

Error prone PCR with dNTP analogues- randomly incorporated instead of the usual dNTPs. Perform normal PCR to amplify mutants.

50
Q

How can error rate be increased in the PCR approach?

A

Imbalance in the amounts of dNTPs or using a non-proofreading polymerase.

51
Q

How can plasmids in E.coli be used for random mutagenesis?

A

Using repair defective E.coli, treat E.coli with chemical to cause DNA damage (e.g. Nitrosguanidine) or use X-Rays/UV to induce mutations.

52
Q

How can DNA shuffling be used to creat mutants?

A

Using DNase I and PCR to produce many variants.

53
Q

How can the mutation rate be increased during DNA shuffling?

A

Perform PCR without primers- means more cycles are required, and more mutations are introduced.

54
Q

How are proteins released from E.coli?

A

Using a sonicator to burst the cells, as the membranes are less robust than in mammalian cells. Freeze-thaw cycles can be used to cause osmotic shock in small volumes of E.coli.

55
Q

In particular, when is recombinant expression absolutely necessary?

A

For expression of membrane proteins for structural analysis. For expression of proteins for NMR analysis as these must be isotropically labelled.

MSF1- Em (7 decks)

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