PCR-mediated expression of the human GM-CSF gene in escherichia coli

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

Cloning and expression of thermus aquaticus DNA polymerase gene, using a thermo - inducible experession vector

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