Lecture 5 Applied Molecular Biology Scanning Mutagenesis - can be done low throughput (small number of mutations) or high throughput Low Throughput . Replace each AA in turn with non-polar such as alanine · Leucine and cysteine are also occasionally used in mutagenesis . Examine effect on protein - is activity abolished, modified or unchanged . In this way key amino acids are identified . Note we can't be certain if a key AA is needed for structure or function . If you find key AA you can manipulate with different AAs · Read about high throughput High throughput mutagenesis - make all 20 AA substitutions at once, at a variety of points in the sequence, ending up with mixture of proteins, mixture called library. Screen the library for a protein with the required activity Protein Library's - made at the genetic levels · By saturation mutagenesis · Or molecular evolutions · Protein libraries contain many variants of just one protein Saturation Mutagenesis . Also know as codon randomisation · Used when you know which amino acids are key to protein activity e.g through scanning mutagenesis · Codons (3x AA) are replaced with randomised codons via cassette mutagenesis . Each Plasmid will pick up different strand of random DNA creating the library . NNM or NNK allows coding of all 20 AAs Play 3 2nd Base A Lys Asn Lys A Asn Gln His C Gln 1st Base His Pro Glu Asp Glu Asp G * T Tyr * Tyr C Thr Thr Thr Thr Pro Pro Pro Ala Ala Ala Ala Ser Ser Ser Ser G T Arg Ser Arg Ser Arg Arg Arg Gly Val Gly Gly . Cys Trp Cys Ile Ile Met Ile Leu Leu Arg Leu Leu Val Gly Val Val Leu Phe Leu Phe NNK-NNK-NNK 3' -NNM NNM NNM 5 Where N = A/C/G/T K = G/T, M = A/C A c G NNN - codes for all 64 codons, But unequal coding's for AAs. E.g. 6x for leucine but only 1x for tryptophan. Also codes for 3x stop codons ending it prematurely T A C G 3'ª Base T A C G 2ºº Base T A A c Lys Asn G A Lys T Asn Gln C His Pro Gln Pro 15 Base His Glu Ala Ala Asp G Glu Asp . Tyr T . G T Arg Ser Arg Ser Arg Arg Arg Arg Gly Gly Va Gly Gly Val Tyr C Thr Thr Th Thr Pro Pro Ala Ala Ser Ser Cys Se Ser Phe T Trp Leu G Cys lle lle Met lle Leu Leu Leu Let Va Val Leu Phe A C G T A C G T 3" Base A C G T A c
NNK - 32 codons for 1 or 2 copy's of each AA. Still all 20 AAs and only 1 stop codon MAX saturation = 1 codon for each AA so 20 codons in total - no degenerate, no extra codons involved Consequences of