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Site-directed Mutagenesis

DNA cloning
Requirements for DNA cloning:
(1) A self-replicating segment of DNA

bacteriophage, plasmid, vector

(2) A procedure to introduce foreign DNA into a functioning cell

transfection, electroporation

(3) A procedure for selecting cells that have incorporated this DNA

antibiotic resistance

Site-directed Mutagenesis
gene insertion

Use of nucleases to cleave DNA at specific recognition sites

Site-directed Mutagenesis
gene insertion
An artificial self-replicating DNA vector Contains a selectable marker (ampicilin resistance)

Contains a polycloning site for gene insertion

Site-directed Mutagenesis
gene cloning procedure
1. Locate specific cleavage sites at the beginning and end of the gene of interest 2. Treat the vector with the same nucleases to create a compatible opening 3. Mix and anneal the vector with the gene 4. Covalently couple the fragments (DNA ligase) 5. Insert into cells to replicate (transformation)

Site-directed Mutagenesis
gene cloning procedure

Site-directed Mutagenesis
gene cloning procedure

1. Plasmids typically contain an antibiotic resistance gene 2. After transformation the bacteria is grown on a plate which contains that antibiotic 3. Only cells that have incorporated the plasmid will be capable of growing to produce colonies

Site-directed Mutagenesis
PCR cloning
Design two sets of primers, one set containing the desired mutation Extend each primer with DNA polymerase

Denature and re-anneal the DNA strands to produce heteroduplexes Only one heteroduplex can be extended from 3 to 5

Site-directed Mutagenesis
Gateway cloning
(1) insert the gene into an entry vector
ccdB attP1
attB1 gene attB2

gene
attP2

attL1
attR1 ccdB attR2

attL2 Entry Clone KanR

Donor Vector KanR

BP Clonase II

(2) transfer the gene to an expression vector


gene attL1 Entry Clone KanR attL2

ccdB attR1 attR2 Destination Vector AmpR


attP1

ccdB attP2 Donor Vector

gene

attB1

attB2 Expression Clone AmpR

LR Clonase II

KanR

Site-directed Mutagenesis
experimental protocol
1. Pick target amino acid to be changed
chemical modification pH studies sequence homology

2. Design a synthetic oligonucleotide to mutate the target amino acid


3. Use this primer to synthesize double stranded DNA 4. Verify production of the mutation 5. Characterize the new enzyme

Site-directed Mutagenesis
verifying the mutation
1. Failure to grow
if this is an essential enzyme for survival of the organism then creating a non-functional mutation should impair survival

2. Gene sequencing
sequence the DNA to verify that the base changes have been introduced

3. Restriction mapping
use the creation of a new restriction site or the elimination of an existing one to verify the mutation

Site-directed Mutagenesis
restriction screening
Allows selection of mutant enzymes through the creation or elimination of a restriction endonuclease site

The creation or elimination of a site changes the size of the DNA fragments obtained

Site-directed Mutagenesis
characterization of mutant enzymes
1. Does the mutation affect any of the properties of the enzyme ?
kinetics substrate binding stability

2. What is the magnitude of the effects ?


does a conservative replacement lead to large effects ? does a less conservative replacement increase the effect ?

3. What conclusions can be drawn about the role of the amino acid that has been mutated ?

Site-directed Mutagenesis
kinetic characterization of mutant enzymes

Conclusions Cys135 provides an important catalytic functional group Gln162 does not appear to be absolutely essential Arg267 plays a role in substrate binding

Site-directed Mutagenesis
Expanded approaches
Three difference experimental approaches have been used to site specifically incorporate unnatural amino acids into enzymes:

1. Peptide ligation
Synthesize a peptide containing an unnatural amino acid and then selectively ligate it between two fragments of the polypeptide

2. Chemical modulation
Use mutagenesis to introduce a reactive amino acid and the selectively modify it with a series of amino acid analogue reagents

3. Supressor mutagenesis
Introduce a stop codon at a specific site and then use a modified tRNA to incorporate unnatural amino acids

references: SynLett 6, 733 (2001) Arch. Bioch. Biophys. 421, 283 (2004) Ann. Rev. Biophys. 24, 435 (1995)

Site-directed Mutagenesis
any amino acid in a protein can be selectively replaced with another amino acid the replacements are made at the genetic level by modifying the codon to incorporate the new amino acid characterizing the mutant enzyme that is obtained will provide information on the role of the amino acid that has been replaced the only unequivocal result from mutagenesis studies is when the mutation has no effect on the enzymes function