Isopentenyl diphosphate isomerase. Site-directed mutagenesis of Cys139 using “counter” PCR amplification of an expression plasmid
✍ Scribed by Ian P. Street; Hazel R. Coffman; C.Dale Poulter
- Book ID
- 104204019
- Publisher
- Elsevier Science
- Year
- 1991
- Tongue
- French
- Weight
- 455 KB
- Volume
- 47
- Category
- Article
- ISSN
- 0040-4020
No coin nor oath required. For personal study only.
✦ Synopsis
Sitedirected mutaQenesfs of Cys139 in yeast isopentenyf diphosphate isomerase was accomplished by "counter" pofymerase chain reactfon ampfiffcatfon of the gene in expression pfasmfd pips241 using only two DNA primers. One primer complemented a region of the sense strand and contained the mutations. The second primer was an exact complement of the antfsense strand. When hybridized to the pfasmid template, the 5'-ends of the primers butted against one another, and PCR amplification resulted in synthesis of double strand linear mutant expression piasmid. The linear DNA was circularized by blunt end ligation to yield an expression plasmid that overproduced mutant yeast enzyme in transformed E. colihosts. Two site-directed mutants, C139A and C 139V, were both inactive, suggestfng mat Cys139 is an essential part of the enzyme's catalytic machinery.