Recombinant Protein Expression - Intro/Bacteria Flashcards Preview

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What is recombinant protein expression?

This is expression (often over-expression) of a protein from a cloned gene in either the same organism or a different organism (homologous/heterologous)


What are some applications of recombinant protein expression?

Diagnostic assays
Industrial enzymes
Drug discovery screening - purified protein/cell-based
Academic and applied research - purified protein/cell-based


What are the scales of recombinant protein expression?

Small, Mid (lab) and industrial
This is dependent on the application


Give the definitions of transfection, transient transfection and stable transfection?

Transfection: The process of introducing a vector into a cultured mammalian (or other higher eukaryote) cell - can be transient or stable

Transient transfection: The vector functions only in the cell that it enters and is not replicated (copied)
It disappears as the host cell divides and the cell population increases - gives a short burst of protein expression over 1-3 days

Stable transfection: The vector permanently integrates into the genome of the host cell after transfection
Detected by screening for co-integration of an antibiotic resistance gene
The capability for protein expression is permanent


What are the different expression systems for a recombinant protein expression experiment?

In Vitro


What are some features of deciding which expression vector to choose?

Is the protein...
Level of activity required?
Soluble or membrane-associated?
Post-translationally modified?
Scale of production required
Ease of transfection
Control required?


Give an overview of the expression systems?

In vitro - good for looking at protein-protein interactions but won't produce any protein
E.coli - go to, it has the capacity to produce large amount of bacterial protein, but not great for expressing eukaryotic genes
Fungal - medium, is relatively easier to grow at a low cost
Insect - Eukaryotic system = good functional protein
Mammalian - small quantities but guaranteed protein expression


What is the actual method of recombinant protein purification?

1. Decide on purification strategy and use this to guide cloning strategy eg addition of fusion tags
2. Clone coding region for target protein into suitable expression vector(s) including any necessary additional sequences
3. Introduce the cDNA encoding the target into the host cell (transform or transfect the host cells)
4. Induce expression of the protein and verify its presence in cells
5. Collect and break open cells or collect medium
6. Purify protein
7. Store protein appropriately for use in assays/structural studies
We don't want the protein to die


What are fusion tags?

Allow easy identification and purification of protein
Some can be useful for improved stability and folding
Fusions may be added either N- or C-terminal and in-frame with the protein coding region
At the N terminus is probably removes start codon
At the C terminus it removes stop codon to create fusion with the tag
PCR normally used to amplify the correct coding region


What is the fastest/simplest way to purify tagged proteins?

Affinity Chromatography
This involves the specific binding of a protein to a ligand immobilised onto a support matrix
Non-tagged proteins pass through and any that do stick non-specifically can be washed off before the recombinant protein is specifically eluted normally using a competitor compound in the elution solution
The competitor can bind to the ligand displacing the protein which can be collected in a pure form


What are some common fusion tags for purification?

Protein - affinity column

Glutathione-s-transferase - glutathione
Maltose binding protein - amylose (from starch)
Hexa-Histidine tag - metals (Ni2+/Co2+)
StrepTag II - StrepTactin


Describe the tags: His, Strep and maltose?

H-H-H-H-H-H –(H-H) coding region added to gene (6 or 8 His)
N- or C-terminal
Load NTA resin with nickel ions (Ni2+)
6-His chelates NICKEL on affinity matrix
Load cell lysate, wash to remove non-specific binding proteins
Elute with imidazole (or strip nickel from column with EDTA)

Strep tag II purification
Collect cells by centrifugation
Lyse by cell disruption/sonication or detergent
Centrifuge and purify from supernatant
Bind to strep-tactin resin
Via streptag - WSHPQREK (mimics biotin)
Elute with competition of desthiobiotin (biotin analogue)

Maltose binding protein
Cloning/expression of target gene
Bind to MBP via a protease cleavage site
Load and wash - amylose binds
Elute with maltose
Cleave with specific protease to release target protein


What can fusion tags be associated with?

Protease cleavage sites


What are some advantages and disadvantages of having E.coli as a host for recombinant protein expression?

Simple & rapid culture
Easy to transform
Well characterised (genome sequence)
Range of vectors/markers

Requires cDNA (no introns)
Lacks much post-translational processing
Possible protein stability/solubility/toxicity issues


Give and overview of recombinant protein expression in E.coli?

Starting with cDNA - we clone the coding region into the expression vector
Using restriction enzymes/ligation, PCR cloning etc...
Make sure it has a promotor in front of it e.g. Tac, T7, pBAD
Ribosome binding site after the promotor and before the start codon
We need to make sure it doesn't form secondary structures
Stop codon at the end or removed and added a fusion tag

May need:
A signal sequence - if the protein needs to be secreted
Di-sulfide bonds
Is the codon usage the same as the host - we may have to change the codons
Needs mRNA stability


Describe the T7 promotor system in E.coli?

Most common in E.coli
It has 2 key elements:
A coding region for T7 RNA polymerase
This is controlled by the LacUV5 promotor
LacI repressor switches off the LacUV5 promotor when the T7 polymerase is not in use - to prevent transcription/translation of RNA

Our gene of interest is only induced due to the host cell being activated so the T7 polymerase binds to the T7 promotor
Cells carrying this genomic T7 RNA polymerase gene insertion are known as DE3 cells eg BL21(DE3)

When we add IPTG, we remove the Lac repressor which binds the inducer
This can allow T7 RNA polymerase production, which can then act on the T7 promotor on the plasmid


How can we optimise codons for E.coli expression?

Some E. coli strains have additional tRNA genes to enhance expression of these genes
Mutate critical codons to more commonly used codons
Resynthesise the complete gene to reflect host codon usage

DNA sequence optimisation does NOT change encoded protein sequence


What is significant about the location of a protein within the expression system?

Default expression location for soluble proteins is in the cytoplasm
But if we want it to be secreted we can add a signal sequence
20-30 amino acids long: basic region, hydrophobic region and cleavage region


What is significant about secondary sturcture within the expression system?

If mRNA has extensive secondary structure - this can prevent access to the ribosome binding site or initiating the start codon
Secondary structure is not usually an issue if expressing an N-terminal fusion protein because the vector will be optimised for expression of the fusion partner
This can be a problem if you have the tag at the C terminus


What is autoinduction?

Grow the cells to appropriate optical density that remains with the IPTG inducer for several hours
We can see what levels of expression we get

We can start by growing them on glucose
Glucose is preferred and no lactose is taken up due to catabolite repression
Small amounts of permease and b-galactoside are expressed - converting lactose into allolactose
Allolactose removes the repressor in the lac promotor
Now there is higher expression of permease and b-galactoside therefore increase lactose coming into the cell and more lactose into allolactose
cAMP increases which associates with catabolite activator protein - allowing RNA polymerase to associate with the promotor = full expression of the lac operon

Glycerol provides an alternative carbon source to allow protein production but does not interfere with lactose system


Give an overview of autoinduction within E.coli?

In autoinduction cells grow on glucose to high cell density
When glucose depleted they start to use lactose which is converted to allolactose and switches on expression of the gene
We can switch on gene expression by adding IPTG (chemical inducer)


Give a summary of E.coli as an expression vector?

Large quantities = good for structural biology work
Reliable for bacterial proteins
Various plasmid vectors and engineered host strains available
Variety of affinity tags to support purification work well in E. coli

To improve yield of protein:
Try different constructs/strains/growth conditions
Use autoinduction

But problematic for:
Proteins of eukaryotic origin -(but not always) not functional
Large multi-domain proteins
Proteins comprising multiple polypeptides
Proteins where post translational modifications are important