June 09, 2009 Random Mutagenesis

Transcription

1 Why Mutagenesis? Analysis of protein function June 09, 2009 Random Mutagenesis Analysis of protein structure Protein engineering Analysis of structure-function relationship Analysis of the catalytic center Design of proteins with novel features Site-directed vs. random How to insert mutations randomly Site-directed Knowledge about sequence / structure Insertion of single mutations possible Number of mutants limited Time-consuming Analysis of highly conserved postions Random Less information about relevant positions Saturating analysis impossible Introduction of more than one mutation Efficient screening system Generation of mutant libraries E. coli XL1red UV irradiation Chemical methods Deamination Alkylation Base-Analog Mutagens PCR based methods DNA shuffling Error prone PCR Site directed random mutagenesis DNA Shuffling PCR based random mutagenesis Slightly different genes coding for the same product Homologous recombination of genes Generation of gene libraries Recombination of sequence blocks In vitro molecular evolution 1

2 DNA Shuffling Parent sequence Denaturization Analysis of large protein regions DNase treatment PCR w/o primers PCR conditions enhance mismatches Saturating mutagenesis virtually impossible Random fragments Combination of mutations Shuffled produkt Extension via polymerase dctp, dttp dgtp, datp Mg 2+ Mg 2+ Mg 2+ stabilizes non-complementary base pairs Mg 2+ Mn 2+ stabilizes non-complementary base pairs reduces the specificity of the polymerase low annealing temperature template amount, cycle number 2

3 Analysis of conserved regions PCR with degenerated primers 5 -CAC ATC AGA ACC NNN GTG TGG GTA AGA-3 -NNN- represents 64 codons 19 possible mutants per position NNN Examples NNN 3

4 Example: DNA shuffling Example: Novel Enzymes Enzyme engineering DNA shuffling Optimized function of Monooxygenase Degradation of pollutants Synthesis of chemicals Ligand binding abilities Conversion of antagonist to agonist No hint, which AA involved Combination of mutations necessary Screening of mutant libraries Error prone PCR Vardar, G and Wood, TK Appl Environ Microbiol Example: Example: Sense primer: N-terminal domain Antisense primer: C-terminal domain 7 mm MgCl 2, 0,5 mm MnCl 2, 1 mm dttp/dctp, 0,2 mm datp/dgtp Screening of about 10 million mutants Efficiant screening system! 13 atropine mutants identified All contained combinations of mutations Glu Trp Asn Pro Identification of essential receptor determinants Mutation of specific AA Single mutations requested Saturating mutagenesis necessary Site-directed random mutagenesis 1 primer -NNN- 64 codons/20 AA Problems: Coverage Wild-type prefered New primer design Exclusion of wild-type Specific for every AA 4

5 1 primer -NNN- 64 codons/20 AA 2 primers for Valine mutation -WTK- -NVK- 28 codons/19 AA 3 primers for Valine mutation -VVG- -WKS- -NAT- 21 codons/19 AA 5