BIOL114 - Lecture 6

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  • Created by: Katherine
  • Created on: 16-03-16 11:35
Why do we need recombinant proteins?
1.To generate large amounts of specific proteins at low cost with high purity. 2.) Where appropriate, to enable these proteins to be modified in ways that improve their properties = protein engineering.
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Why might this be important?
Because many proteins are expressed at very low levels. Availablity of the the source tissue may be very limited. Source tissue can potentially be contaminated.
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Give an example of a therapeutic protein:
Human insulin - it is much better to produce a human recombinant treatment then use animals.
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Give 4 examples of recombinant therapeutic proteins:
Insulin, Human growth hormone, Factor viii, tPA
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How can recombinant proteins be expressed in E.coli?
1.) Expression vectors (plasmids designed for transcribing and translating a cloned cDNA insert.
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What are the methods for improving levels of expression?
1.) inducible promoter (e.g. lac promoter), codon usage optimization (to improve rates of translation), N terminal fusions (to improve rates of translation and protein stability)
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What are the methods of facilitate protein purification?
Fusion tags (for affinity purification), Signal peptides (for secretion)
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What makes an expression vector different from a normal vector?
Origin of Replication, selectable marker,
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What is important about an exmpression vector?
It has the necessary sequences that allow the inserted cDNA sequence to be both transcribed and translated. So transcription is driven by the promoter region and there is a ribosome binding site where translation occurs.
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A cDNA which you've taken from a Eukaryotic cell will not have
a ribosomal binding site.
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The expression vector also has a... because this is not found on the cDNA.
Terminator of transcription
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In order for an expression vector to work, what must be the case:
Inserted DNA must be cDNA (no introns), Inserted cDNA must be adjacent to appropriate sequences for its transcription and translation in the host cell. Must be a high copy number plasmid.
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What are the methods for improving levels of expression?
Inducible promoter?
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What does inducible mean?
You can switch it on when you want it on.
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Why would you want an inducible promoter?
Allow the expression of the recombinant protein to be switched on only when the bacterial culture has become established.
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Why are inducible promoters advantageous?
Because recombinant proteins are often toxic for the bacteria and slow frowth - so there is strong selection pressure for elimination and rearrangement of the plasmid.
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Which inducible promoter is most frequently used?
lac promoter - regulates transcription of the lac operon
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How does the lac operon work?
When lactose is absent, the repressor is active and lac operon is off., so no RNA is made.
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What happens when lactose is present?
The repressor is inactive, lac operon is on and therefore mRNA is made.
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What is IPTG?
A stable analogue of lactose which binds to the Lac repressor protein. It is an effective inducer.. Lac repressor IPTG complex no longer binds to lac operator. Addition of IPTG to culture induces expression from the lac promoter.
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What are the methods of improving expression?
Optimising codon usage. There are distinct idfference between codon usage in E.coli and humans that are refleted in the abundance of tRNAs, making translation of human sequences inefficient.
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Codon optimization strategies -
Chemically synthesize new gene - alter sequence of the gene of interest to match donor codons to the codons most frequently used in host organisms.
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How many codon optimization strategies are there?
2
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What are the two codon optimization strategies?
Chemically synthesize new gene and use and engineered host cell that over expresses low abundance tRNAs
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Why are N terminal fusions good?
They allow higher expression levels (rates of translation), increase stability of expressed protein - particularly if proteins are small, also used to facilitate purification of the protein - fusion tags.
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The expression should have:
Protease cleavage site to allow the N terminal fusion to be removed later if necessary, multiple cloning site to facilitate insertion of the cDNA encoding the protein of interest.
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After what can the N terminal be removed?
After affinity purification it can be removed if it is linked to the rest of the protein by a suitable protease target sequence e.g. thrombin.
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What are the methods to facilitate protein purification?
Fusion tags (for affinity purification), signal peptides (for secretion)
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What is a fusion tag?
A specific type of N terminal fusion designed to make subsequent purification of the recombinant protein easier.
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Give an example of a fusion tag:
A polyhistidine tag allows affinity purification on an immobilised nickel chromatography column.
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Give two more examples of fusion tags:
MBP, Gluthione S-Transferase
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Why can purification sometimes be difficult?
Expression of large amounts of foreign proteins in E.coli results in protein precipitating out.
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What is a signal peptide used for?
To target bacterial proteins out of the cell, used to export a foreign protein to the periplasm.
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How many domains does a signal peptide have?
3
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The signal peptide is removed by what?
Host proteases as it is exported.
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This methodology is particulalry applicable to proteins that are normally secreted in what
Eukaryotes
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During the first decades of insulin therapy, what insulin had to be used?
Bovine or porcine.
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What are the advantages of recombinant human insulin?
It can be produced in limitless quantities, chaply. It is identical to native insulin and therefore less likely to provoke an immune response.
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How many polypeptide chains does insulin consist of, and what are they connected by?
2, Disulphide bonds
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Sometimes it is necessary to modify the properties of the recombinant protein . e.g. it's...
Stability and kinetics.
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How is the stability of the recombinant protein modified?
Increased thermostability, increased stability at low/high pH, resistance to oxidative inactivation (for longer shelf life)
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How are the kinetics of the recombinant protein modified?
Increased affinity for substrate, increased speed of reaction - altered substrate specificity.
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Examples of protein engineering include:
tPA - tissue plasminogen activator.
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How has recombinant tPA been engineered?
To have increase in vivo stability
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Examples of protein engineering include:
Subtilisin - alkaline protease from Bacillus subtilis
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How has subtilisin been engineered?
To be stable at 70'C, resistant to non-ionic detergents, resistant to oxidation (for longer shelf life)
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What is Psedomonas syringae?
It is a bacterium that colonises plant surfaces. Some strains contain a membrane proein known as ice nucleation protein (INP) that acts as a template for the formation of ice crystals.
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How can microorganisms be used to convert one organic compound into another?
Synthesis of theraputic steroids from diosgenin.
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What is bioremediation?
The process of reclaiming or cleaning up contaminated sites using microorganisms to remove or degrate toxic wastes. Used in sewage treatment, chemical degradation in soils, bioremediation of oil spills.
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What happens during the bioremediation of oil spills?
Spraying of fertilizer to stimulate growth of indigenous bacteria that can breakdown the oil - fastest and cheapest way to clean up beaches.
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What are three elements needed adjacent to a cDNA for expression in a bacterial plasmid?
Promoter, transcription terminator, Ribosome binding sequence.
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What are three therapeutic recombinant proteins?
Human insulin, Erythropoietin, tPA
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What are three reasons for expressing recombinant proteins as fusion proteins?
Increased expression, simpler purification, greater stability
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What are 3 fusion tags?
Polyhistidine, Glutathione-S-transferase, Maltose Binding protein
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What are three ligans used in purifying fusion proteins?
Nickel, Amylose, Gluthathione
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What is a protein sequence used to direct protein secretion?
Signal peptide
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What is an enzyme used in biological washing powder?
Subtilisin
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What is an Ice-nucleating bacterium?
Pseudomonas syringae
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What is a precursor for steroid production by microbial biotransformation?
Diosgenin
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Explain why cDNA is ued to express eukaryotic proteins in bacteria
f
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List the features of typical bacterial expression vectors
f
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List examples of medically proteins epressed in E.coli
J
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Describe how insulin is produced from E.coli
f
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Explain the various reasons why foreign proteins may be epressed as fusion proteins in E.coli
j
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Describe the features of vectors used for expression of fusion proteins
f
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List different fusion tags and their uses
d
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List the characteristics of recombinant proteins that may be improved by protein engineering
f
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Give examples of engineered proteins
g
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Discuss other (non recombinant) uses of microbial biotechnology
f
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Define the terms: Fusion protein, His-tag, Bacterial signal peptide, Subtilisin, tPA, Thrombin, Bioremediation, Bioconversion, Protein engineering, SSite directed mutagenesis
g
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Card 2

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Why might this be important?

Back

Because many proteins are expressed at very low levels. Availablity of the the source tissue may be very limited. Source tissue can potentially be contaminated.

Card 3

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Give an example of a therapeutic protein:

Back

Preview of the front of card 3

Card 4

Front

Give 4 examples of recombinant therapeutic proteins:

Back

Preview of the front of card 4

Card 5

Front

How can recombinant proteins be expressed in E.coli?

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