Protein Engineering Flashcards

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1
Q

Uses of protein engineering

A

• Probe mechanism to make changes to molecule to probe how it works
• Create novel proteins for biotech
• Improve catalytic function (kcat or kcat/Km) – can be useful for biotech
• Alter substrate specificity or Stereospecificity – can broaden specificity
• Improve stability (integral membrane proteins are really unstable)
• Requirements growing as biotech applications increase
• More eco-friendly production processes
• Dealing with ecological challenges e.g. plastic and other contamination issues

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2
Q

What techniques can be used to generate mutants

A

• Synthetic gene route
• Plasmid based approach
• Overlap extension methods using PCR
• Alanine scanning mutagenesis
• Random mutagenesis

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3
Q

Synthetic gene route

A

• Good if you want to make lots of single point mutations at the same time
• Good for adding lots of tags and protease cleavage sites
• Generate electronic file of gene with sequence you want to have made and send it off
• They make long oligonucleotide templates and generate the dna

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4
Q

Plasmid based approach

A

• Gene in plasmid has target site for mutation – identify the specific site you want to change
• Denature the plasmid and anneal the oligonucleotide primers containing the desired mutation and substantial regions of dna flanking it
• Using the nonstrand-displacing action of pfuTurbo dna pol, extend and incorporate the mutagenic primers resulting in nicked circular strands
• Need to se a polymerase with proofreading activity
• Long piece of dna —> high chance for error
• PCR generated daughter strands have mutation
• Digest the methylated, non-mutated parental dna template with DpnI
• DpnI wont digest the daughter strand
• Transform the circular, nicked dsDNA into super competent cells
• After transformation the cells repair the nicks in the mutated plasmid

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5
Q

Overlap extension methods using PCR

A

• Need 2 different RE sites
• More complex but can use if plasmid based approach fails
• Need to identify the specific site you want to mutate
• Primers contain the mutation and dna either side
• 2 separate PCR reactions

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6
Q

Alanine scanning mutagenesis

A

• Systematic approach to engineering proteins
• Used to investigate role of residues
• Obtain a more stable construct
• Every residue is mutated to Ala (except in case of Ala-mutated to Leu)
• Can do Ala scan in predefined region of protein
• Start to understand the precise role that each aa has within a protein
• Need to have means of assessing effects of mutations e.g. functional assay
• Depends what you have done to molecule/ are assessing

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7
Q

What is random mutagenesis

A

When you dont have control of where substitutions are introduced

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8
Q

What is chemical mutagenesis

A

• Plasmid treated with chemical
• E.g. sodium bisulfite, nitrous acid, hydrazine dimethyl sulphate
• Induces damage
• Gene can then be amplified by PCR and cloned

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9
Q

PCR approaches for random mutagenesis

A

• Sloppy PCR or error prone PCR (ep PCR)
• Can use non-proofreading pol and change conditions
• Error rate of PCR reaction is deliberately increased
• E.g. increasing levels of Mn2+ or having an imbalance in [dNTP] e.g. by specifically increasing [dGTP]
• Rate of mutation is 0.6-2%

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10
Q

Random mutagenesis of plasmids in e.coli cells

A

• Treat with chemical
• E.g. nitrosoguanidine, ethane methyl solfonate (EMS) or UV/ X rays
• Induces mutations
• Increases level of variation within the plasmids
• Propagate plasmid in repair-defective strains of e.coli
• Defective in repair mechanisms

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11
Q

DNA shuffling: method for in vitro recombination

A

• Fragment sequences with DNAseI
• Purify
• Purify dna samples and use a range of dna templates with single point mutations
• Random piecing together of bits of dna
• A variety of sophisticated methods are now available to shuffle dna
• Proof of principle with gene encoding LacZ alpha
• Can do with or without primers
• Designing primers means you have biased 5’ and 3’ end
• However, full length of dna is regained faster if you use primers

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12
Q

Iterative process of random mutagenesis

A

All mutant genes are expressed and tested
Selection of functional mutants enzymes
Isolation of genes for improved enzymes
Repeat

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13
Q

Screening for variants

A

• Time consuming and can involve addition steps
• Altered substrate isn’t ideal – you alter what you’re screening for as the substrate is different (because of fluorescent tag)
• There are less direct methods
Can do standard substrate conversion
Or chromo/fluorogenic surrogate substrate
Can use chromo/fluorogenic cofactor/cosubstrate
Usually best to use a coupled consecutive signalling reaction (substrate —> product —> product 2) second conversion uses chromogenic cofactor/co-substrate

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14
Q

Iterative saturation mutagenesis (ISM) for rapid directed evolution

A

• Make lots of variants for a specific region
• Used for improved protein function
• Select mutant that performed best then further mutate it with another mutation that showed promise
• Continue until you have the optimal protein
• You make individual substitutions at specific sites

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15
Q

What is UapA

A

• UapA
• Uric-acid xanthine transporter from aspergillus nidulans
• High affinity, high capacity H+ symporter (couples substrate movement with H+)
• Potential anti fungal drug target
• Exceptionally well studied protein
• Express in S. cerevisiae as a fusion with GFP
• Functional assay available so you can check it can still carry out function
• We want a sufficient quantity for structural analysis with x ray crystallography
• No homologue in S. cerevisiae
• Can do functional analysis in cells and know that activity is from UapA and not a homologue

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16
Q

Stability of UapA

A

After 1 day protein begins to degrade
• Did 6 point mutations
• Put wild type down size exclusion chromatography column to track gfp
• Introduced the 6 point mutations then DDM solubilised protein is heated at 45 deg C for 10 mins
• Can see which mutation increases stability vs the wild type
• Mutation with best stability was G411V
• Protein has lost function and is therefore more stable
• If we had used an A there would have been more activity so it would have been more unstable
• React with 1mM Xanthine which is a more harsh detergent and will cause stress
• Long term stability of the protein is also improved

17
Q

Truncation of UapA

A

• Look for regions of the protein likely to be disordered
• Loop regions near the N and C termini
• Generate truncations at N and C termini
• Combine the mutation and the truncation