Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering.
An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.
D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000235:genetics; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D007612:Kanamycin / Q000494:pharmacology; D009154:Mutation; D010957:Plasmids; D001618:beta-Lactamases / Q000235:genetics
D004277:DNA, Single-Stranded; D007612:Kanamycin; D001618:beta-Lactamases