SiteSite-directed Mutagenesis

Mutagenesis gives us the capability of testing the role of any amino acid in a protein by replacing it with any of the other naturally occurring amino acids

electroporation (3) A procedure for selecting cells that have incorporated this DNA antibiotic resistance . plasmid. vector (2) A procedure to introduce foreign DNA into a functioning cell transfection.SiteSite-directed Mutagenesis DNA cloning Requirements for DNA cloning: (1) A self-replicating segment of DNA bacteriophage.

SiteSite-directed Mutagenesis gene insertion Use of nucleases to cleave DNA at specific recognition sites .

SiteSite-directed Mutagenesis gene insertion An artificial self-replicating DNA vector Contains a selectable marker (ampicilin resistance) Contains a polycloning site for gene insertion .

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

SiteSite-directed Mutagenesis gene cloning procedure .

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 . Plasmids typically contain an antibiotic resistance gene 2.SiteSite-directed Mutagenesis gene cloning procedure 1.

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¶ .SiteSite-directed Mutagenesis PCR cloning Design two sets of primers.

SiteSite-directed Mutagenesis Gateway© cloning (1) insert the gene into an entry vector ccdB attB1 gene attB2 attR gene ccdB attR attP1 attP2 Donor Vector KanR attL (2) transfer the gene to an expression vector gene KanR AmpR LR ClonaseŒ II KanR AmpR ¦ Entry Clone + Destination Vector + Expressi n Cl ne ¦ © ¨ © ¨ attL attL Donor Vector ©§ ¨§ attR attR © ¨ attP § § ccd ccd attP att ¥ ¤£ BP ClonaseŒ II gene ¢ + + Entry Cl ne n att ¡   attL ¡   .

Design a synthetic oligonucleotide to mutate the target amino acid 3.SiteSite-directed Mutagenesis experimental protocol 1. Verify production of the mutation 5. Pick target amino acid to be changed chemical modification pH studies sequence homology 2. Characterize the new enzyme . Use this primer to synthesize double stranded DNA 4.

Restriction mapping use the creation of a new restriction site or the elimination of an existing one to verify the mutation . Gene sequencing sequence the DNA to verify that the base changes have been introduced 3. Failure to grow if this is an essential enzyme for survival of the organism then creating a non-functional mutation should impair survival 2.SiteSite-directed Mutagenesis verifying the mutation 1.

SiteSite-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 .

Does the mutation affect any of the properties of the enzyme ? kinetics substrate binding stability 2.SiteSite-directed Mutagenesis characterization of mutant enzymes 1. What conclusions can be drawn about the role of the amino acid that has been mutated ? . What is the magnitude of the effects ? does a conservative replacement lead to large effects ? does a less conservative replacement increase the effect ? 3.

SiteSite-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 .

733 (2001) Arch. Rev. 435 (1995) . Supressor mutagenesis Introduce a stop codon at a specific site and then use a modified tRNA to incorporate unnatural amino acids references: SynLett 6. Peptide ligation Synthesize a peptide containing an unnatural amino acid and then selectively ligate it between two fragments of the polypeptide 2. Bioch.SiteSite-directed Mutagenesis Expanded approaches Three difference experimental approaches have been used to site specifically incorporate unnatural amino acids into enzymes: 1. Chemical modulation Use mutagenesis to introduce a reactive amino acid and the selectively modify it with a series of amino acid analogue reagents 3. 24. Biophys. Biophys. 283 (2004) Ann. 421.

SiteSite-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 enzyme¶s function .