Study and engineering of gene function: mutagenesis I. Why mutagenize? II. Random mutagenesis,...
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Transcript of Study and engineering of gene function: mutagenesis I. Why mutagenize? II. Random mutagenesis,...
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Study and engineering of gene function: mutagenesis
I. Why mutagenize?II. Random mutagenesis, mutant
selection schemesIII. Site-directed mutagenesis,
deletion mutagenesisIV. Engineering of proteinsV. Alterations in the genetic code
Course Packet: #30
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Uses for mutagenesis
• Define the role of a gene--are phenotypes altered by mutations?
• Determine functionally important regions of a gene (in vivo or in vitro)
• Improve or change the function of a gene product
• Investigate functions of non-genes, eg. DNA regions important for regulation
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Protein engineering-Why?
• Enhance stability/function under new conditions– temperature, pH, organic/aqueous
solvent, [salt]• Alter enzyme substrate specificity• Enhance enzymatic rate• Alter epitope binding properties
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Enzymes: Biotech Cash Crops
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From Koeller and Wang, “enzymes for chemical synthesis”, Nature 409, 232 - 240 (2001)
Obtaining useful enzymes
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Random mutagenesis
• Cassette mutagenesis with “doped”oligos
• Chemical mutagenesis– expose short piece of DNA to mutagen,
make “library” of clones, test for phenotypes
• PCR mutagenesis by base misincorporation– Include Mn2+ in reaction– Reduce concentration of one dNTP
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Random mutagenesis by PCR: the Green Fluorescent Protein
Screen mutants
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Cassette mutagenesis (semi-random)
Strands synthesized individually, then annealed
Allows random insertion of any amino acid at defined positions
Translation of sequence
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Random and semi-random mutagenesis: directed evolution
• Mutagenize existing protein, eg. error-prone PCR, doped oligo cassette mutagenesis
-- and/or --Do “gene shuffling”(Creates Library)
• Screen library of mutations for proteins with altered properties– Standard screening: 10,000 - 100,000
mutants– Phage display: 109 mutants
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Gene shuffling: “sexual PCR”
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Gene shuffling
For gene shuffling protocols you must have related genes in original pool: 1) evolutionary variants, or 2) variants mutated in vitro
Shuffling allows rapid scanning through sequence space:faster than doing multiple rounds of random mutagenesis and screening
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Shuffling of one gene mutagenized in two ways
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Gene shuffling--cephalosporinase from 4 bacteria
Single gene mutagenesis
Multiple gene shuffling
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Screening by phage display: create library of mutant proteins fused to M13
gene III
Human growth hormone: want to generate variants that bind to hGH receptor more tightly
Random mutagenesis
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Phage display:production of recombinant phage
The “display”
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Phage display: collect tight-binding phage
The selection
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Animation of phage display
http://www.dyax.com/discovery/phagedisplay.html
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Site-directed mutagenesis:
primer extension method
Drawbacks:
-- both mutant and wild type versions of the gene are made following transfection--lots of screening required, or tricks required to prevent replication of wild type strand
-- requires single-stranded, circular template DNA
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Alternative primer extension mutagenesis techniques
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“QuikChangeTM” protocol
Advantage: can use plasmid (double-stranded) DNA
Destroys the template DNA (DNA has to come from dam+ host
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Site-directed mutagenesis: Mega-primer
method
Megaprimer needs to be purified prior to PCR 2
Allows placement of mutation anywhere in a piece of DNA
A
B
Wild type template
First PCR
Second PCR
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Domain swapping using “megaprimers” (overlapping PCR)
N- -C
Mega-primer
PCR 1
PCR 2
Domains have been swapped
Template 1
Template 2
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PCR-mediated deletion mutagenesis
Target DNA
PCR products
Oligonucleotide design allows precision in deletion positions
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Directed mutagenesis
• Make changes in amino acid sequence based on rational decisions
• Structure known? Mutate amino acids in any part of protein thought to influence activity/stability/solubility etc.
• Protein with multiple family members? Mutate desired protein in positions that bring it closer to another family member with desired properties
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An example of directed mutagenesis
T4 lysozyme: structure known
Can it be made more stable by the addition of pairs of cysteine residues (allowing disulfide bridges to form?) without altering activity of the protein?
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T4 lysozyme: a model for stability studies
Cysteines were added to areas of the protein in close proximity--disulfide bridges could form
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More disulfides, greater stabilization at high T
Bottom of bar: melting temperature under reducing condtions
Top of bar: Melting temperature under oxidizing conditions
Green bars: if the effects of individual S-S bonds were added together
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Stability can be increased - but there can be a cost in activity
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The genetic code• 61 sense codons, 3 non-sense (stop) codons• 20 amino acids
• Other amino acids, some in the cell (as precursors to other amino acids), but very rarely have any been added to the genetic code in a living system
• Is it possible to add new amino acids to the code?• Yes...sort of
Wang et al. (2001) “Expanding the genetic code” Science 292, p. 498.
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Altering the genetic code
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Why add new amino acids to proteins?
• New amino acid = new functional group• Alter or enhance protein function
(rational design)• Chemically modify protein following
synthesis (chemical derivitization)– Probe protein structure, function– Modify protein in vivo, add labels and
monitor protein localization, movement, dynamics in living cells
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How to modify genetic code?Adding new amino acids to the code--must bypass
the fidelity mechanisms that have evolved to prevent this from occurring
2 key mechanisms of fidelity
• Correct amino acid inserted by ribosome through interactions between tRNA anti-codon and mRNA codon of the mRNA in the ribosome
• Specific tRNA charged with correct amino acid because of high specificity of tRNA synthetase interaction
• Add new tRNA, add new tRNA synthetase
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tRNA charging and usage
Charging: (tRNA + amino acid + amino acyl-tRNA synthetase)
Translation:(tRNA-aa + codon/anticodon interaction + ribosome)
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• Chose tRNAtyr, and the tRNAtyr synthetase (mTyrRS) from an archaean (M.jannaschii)--no cross-reactivity with E. coli tRNAtyr and synthetase
• Mutate m-tRNAtyr to recognize stop codon (UAG) on mRNA
• Mutate m-TyrRS at 5 positions near the tyrosine binding site by doped oligonucleotide random mutagenesis
• Obtain mutants that can insert O-methyl-L-tyrosine at any UAG codon
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Outcome
• Strategy allows site specific insertion of new amino acid--just design protein to have UAG stop codon where you’d like the new amino acid to go
• Transform engineered E. coli with plasmid containing the engineered gene
• Feed cells O-methyl tyrosine to get synthesis of full length gene
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Utility of strategy
• Several new amino acids have been added to the E. coli code in this way, including phenyalanine derivatives with keto groups, which can be modified by hydrazide-containing fluorescent dyes in vivo– Useful for tracking protein localization,
movement, and dynamics in the cell
p-acetyl-L-phenylalanine
m-acetyl-L-phenylalanine
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Some questions:
• What are the consequences for the cell with an expanded code?
• Do new amino acids confer any kind of evolutionary advantage to organisms that have them? (assuming they get a ready supply of the new amino acid…)
• Why do cells have/need 3 stop codons????