ABSTRACT
Four exons of the human genomic GM-CSF gene were assembled together using gene splicing by overlap extension [SOE] method. The resulting nucleotide sequence was cloned in the pET23a [+] expression vector under the control of strong bacteriophage T7 transcription and translation signals. The construct obtained was Transferred into the E. coli strain, BL21 [DE3] pLysS and IPTG was used for induction of GM-CSF gene and production of the target protein, one mg of protein per liter of cell culture, was obtained as revealed by ELISA
Subject(s)
Escherichia coli , Polymerase Chain Reaction , Gene ExpressionABSTRACT
DNA polymerase gene from Thermus aquaticus strain YT1 was amplified using VENTTH DNA polymerase and cloned under the control of lambda pr promoter and expression was induced by a shift in temperature. The culture was then sonicated, and after centrifugation the lysate was treated with polyethyleneimine followed by a salting-out step. Finally the protein was precipitated with ammonium sulfate and fractionated by gel filtration. The resulting enzyme preparation was stable and active