Expressão de fosfatase alcalina em Escherichia coli sob o controle do sistema de regulação do operon lac

Abstract

Alkaline phosphatase function is to catalyze the hydrolysis of a phosphomonoester, and thus has the property of removing phosphate groups 5' DNA and RNA, making it an important tool for genetic engineering. Expression of the E. coli alkaline phosphatase gene was obtained by cloning the gene into plasmid pBR322. Subsequently phoA structural gene was transferred to the vector pAC92 derivative of pUC18 where its expression was regulated partly yielding the recombinant plasmid capable of programming in E. coli highest level of expression. But this vector has the need to control the expression with glucose medium, otherwise the excess of the enzyme in the periplasm causes cell death of the host. In work carried out in the Laboratory of Technologies of DNA called a vector UFAM Pula was built from obtaining pAC92 promoter region, restored operator, cloned in a vector called pUN, this being the modified pUC18 lacking the gene for β - galactosidase and without its promoter region / operator of origin. This paper describes the expression of the alkaline phosphatase of E. coli by means of the adjustable jumps expression system and purification of this enzyme. Thus, the phoA gene was subcloned into the vector jumps successfully, yielding the vector pUNF, which appeared stable and functional, regulated by the induction of IPTG. The best expression of the enzyme in the system in question occurred in the middle with a concentration of 0.05 % glucose, with 1 mM IPTG and after 18 hours of induction.