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Biotechnology > Biotechnology 6 > Flashcards

Biotechnology 6 Flashcards

1
Q

Why do we need recombinant proteins?

A

Mass production

Protein manipulation

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2
Q

What is a therapeutic use of recombinant proteins?

A

Insulin
tPN
EPO

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3
Q

What are 3 important consideration when choosing expression systems?

A

Size of target protein
Solubility of target proteins
Post translational modifications

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4
Q

Why is E Coli used as a common host cell?

A 
Simple 
Cheap 
Fast grow
High yield 
Genetics
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5
Q

What components can be optimised with E coli?

A

The E coli strain
The plasmid vector
The coding cDNA sequence inserted

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6
Q

What are the essential components of expression plasmid design?

A 
Promoter
`ribosome binding sequence
Cloning site
Transcription terminator 
Selection gene 
Replication origin
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7
Q

What are the optional components of expression plasmid design?

A 
N terminal sequence 
Signal peptide for secretion
Fusion tag for purification
PolyA signal
Chaperone encoding gene
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8
Q

What is lac promoter used for?

A

Inducible promoter

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9
Q

What does a lac promoter do?

A

Regulates transcription of the lac operon

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10
Q

What happen sand lactose is absent (the lac promoter)?

A

Repressor is active and lac operon is off

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11
Q

What can the lac promoter be used for?

A

Improve protein yield

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12
Q

Define inducible promoter

A

Allow the expression of the recombinant protein to be switched on only when the bacterial culture has been established

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13
Q

Why are inducible promoters desirable?

A

Recombinant proteins are often toxic for bacteria and cause slow growth which doesn’t occur with inducible promoters

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14
Q

Why do codons need to be optimised?

A

There are differences between codon usage in E coli and humans
therefore corresponding tRNAs are not found in the same proportion

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15
Q

Identify 2 means of optimising codon usage

A

Replace human favoured codons for bacterial favoured ones by chemical cDNA synthesis and site-directed mutagenesis
Use modified E. coli strains overexpressing rare tRNAs

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16
Q

What are benefits of incorporating N terminal fusions?

A

Can improve translation rate
Can increase protein stability
Can facilitate purification

17
Q

Define fusion tag

A

A specific type of C or N terminal fusion designed to facilitate subsequent purification of recombinant proteins

18
Q

When can a N terminal fusion be removed?

A

After purification if it linked to the rest of the protein by a suitable protease target sequence

19
Q

What is a polyhistidine tag?

A

For affinity purification on an immobilised nickel chromatography column

20
Q

Give 2 examples of fusion tags

A

MBP

GST

21
Q

What is an advantage of inducing recombinant protein secretion?

A

E. coli cytosol is not favourable to foreign protein folding whereas its periplasm is
Large amounts of intracellular proteins can form aggregates
To improve protein solubility and facilitate harvesting

22
Q

What are the benefits of adding N terminal signal peptide?

A

In periplasm host proteases can cleave the signal peptide
Periplasmic host enzymes also assist protein folding
Osmotic shock is sufficient to release recombinant proteins (no cell lysis)
It can improve protein stability and solubility

23
Q

Why is recombinant human insulin used?

A

Cheap
Limitless quantities
Non-immunogen

24
Q

How is recombinant human insulin produced?

A

A & B chains fused to β-galactosidase for stability
Transformed separately in 2 different E. coli
Affinity-purified with column/beads bound to β-galactosidase ligand
Cleavage of β-galactosidase with cyanogen bromide
A & B mixed, refolded, cysteine oxidized to form S-S bonds

25
Q

What are 3 reasons for protein engineering?

A

Improve protein stability
Modify protein kinetics
Create something new

26
Q

What are 2 requirements of protein engineering?

A

Detailed knowledge of protein

Site-directed mutagenesis or chemical gene synthesis

27
Q

What are 3 examples of protein engineering?

A

Fluorescent proteins
tPA
Subtilisin from Bacillus subtilis

28
Q

Name 4 examples of natural microbial compounds

A

Flavouring compounds
Organic solvents and acids
Secondary metabolites
Polysaccharides

29
Q

Define bioremediation

A

Reclaiming or cleaning up contaminated sites using microorganisms to remove or degrade toxic wastes.

30
Q

Give 2 applications of bioremediation

A

Oil spills

Chemical degradation in soils

Biotechnology (10 decks)

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