Protein Engineering Flashcards
Uses of protein engineering
• 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
What techniques can be used to generate mutants
• Synthetic gene route
• Plasmid based approach
• Overlap extension methods using PCR
• Alanine scanning mutagenesis
• Random mutagenesis
Synthetic gene route
• 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
Plasmid based approach
• 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
Overlap extension methods using PCR
• 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
Alanine scanning mutagenesis
• 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
What is random mutagenesis
When you dont have control of where substitutions are introduced
What is chemical mutagenesis
• Plasmid treated with chemical
• E.g. sodium bisulfite, nitrous acid, hydrazine dimethyl sulphate
• Induces damage
• Gene can then be amplified by PCR and cloned
PCR approaches for random mutagenesis
• 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%
Random mutagenesis of plasmids in e.coli cells
• 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
DNA shuffling: method for in vitro recombination
• 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
Iterative process of random mutagenesis
All mutant genes are expressed and tested
Selection of functional mutants enzymes
Isolation of genes for improved enzymes
Repeat
Screening for variants
• 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
Iterative saturation mutagenesis (ISM) for rapid directed evolution
• 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
What is UapA
• 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
