1. 1. The cDNA gene coding for the enzyme transglutiminase (EC 18.104.22.168) was cloned into the pUC18 oriented for expression from the lac promoter. 2. 2. DNA sequencing of the 5' end showed that the cDNA was missing the sequence coding of the N-tenninal 30 amino acids. 3. 3. The truncated gene was then cloned into pKK.233-2, and the recombinant product was produced in Escherichia coli. 4. 4. A gene construct coding for the complete protein was generated by inserting an oligonucleotide for the missing 30 amino acids into the Eco RI site of the pUC18 clone. 5. 5. A consensus Shine-Dalgamo sequence and translational start codon were positioned at the 5' end of the linker. 6. 6. Immunoblotting experiments of E.coli JM105(pUC18-TGase) indicated the expression of the transglutaminase gene. 7. 7. The cell lysate as well as the partially purified transglutaminase showed no detectable enzyme activity. © 1991.
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