Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function.
We have developed a foolproof method to substitute a stretch of residues by alanines. After the introduction of aPstI site by IPCR, thus creating two alanine codons, additional codons are introduced at this site through the use of an 'alanine-stretch cartridge'. These cartridges comprise an antibiotic resistance gene flanked on both sides by alanine codons. Excision of the resistance gene byPvuII then yields the correct insertion of codons. The method is both highly reliable and flexible and should be of general use.
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Institut de Pharmacologie et Biologie Structurale, 205 Route de Narbonne, F-31077 Toulouse Cédex, France.
Rémy
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MH
Masson
J M
JM
D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D000667:Ampicillin / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D003062:Codon / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D011506:Proteins / Q000737:chemistry*; D014170:Transformation, Genetic / Q000235:genetics
D003062:Codon; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D002701:Chloramphenicol; D000667:Ampicillin; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D000409:Alanine