Using polymerase chain reaction (PCR) technique we have selectively amplified two-thirds of E coli DNA polymerase I gene from the genomic DNA corresponding to the coding region of proteolytically derived 'Klenow fragment'. The PCR product was cloned in the E. coli expression vector pET-3a. Transcription of Klenow gene fragment in this clone was initiated from T7 RNA polymerase promoter, whereas translation was driven by the Shine-Dalgarno sequence and the initiator AUG cod on of the T7 gene 10 message. The clone was introduced into an appropriate E. coli strain in which T7 RNA polymerase was expressed under the control of the lac promoter. Under optimal conditions of induction with tsopropylthiogalactopyranoside (IPTG), Klenow polypeptide made in this bacterial strain constituted 10-15% of total cellular protein. Klenow potypeptide has been purified to homogeneity in a single step by immunoaffinity column chromatography. The purified Klenow fragment showed identical specific activity as the commercially available product The availability of such a clone will be greatly helpful in carrying out site-directed mutagenesis to examine the regions on the enzyme molecule which are implicated in the DNA synthesis.
|Original language||English (US)|
|Number of pages||6|
|State||Published - Jan 1 1993|
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