Question

Expressing eukaryotic genes in E. coli: Describe important steps (include various enzymes and their functions) needed to clone and express proinsulin in E. coli (exclude purification of the protein)

Answer 1

Initially the gene of interest insulin has to be attached to a plasmid using restriction enzyme and ligase enzyme and it has to be transferred into e coli cell and the enzymes needed for the synthesis eukaryotic gene expression and post translational modification enzymes also can be cloned to avoid the post translational errors .

Initiallybwe have to amplify insulin gene using PCR and restriction digestion of both plasmid and gene using same enzymes and ligation using ligase enzyme and e coli transformation using heat shock method and than selction of recombjnants and growing in large scale