CONSTRUCTION OF HUMAN GM-CSF GENE USING POLYMERASE CHAIN-REACTION ANDITS EXPRESSION IN PSEUDOMONAS-PUTIDA CELLS

Human GM-CSF gene was constructed using polymerase chain reaction (PCR ). Four exons of the GM-CSF gene were amplified using thermostable Tth DNA polymerase on the basis of genomic DNA extracted from human venou s blood. Synthetic oligonucleotides containing sequences complementary to the exon ends were used as primers. The exons were joined by recip rocal complementation of the terminal sequences and subsequent amplifi cation of the joined products. In most cases, effective synthesis of e xons and their junction products was possible only after optimization of PCR conditions for each primer pair. These procedures resulted in c reation of a GM-CSF gene consisting of four exons. The nucleotide sequ ence analysis of the synthetic gene showed its complete identity with the natural one. The gene was inserted into an expression vector under control of the P-tac-P-lac promoter tandem Expression of the GM-CSF g ene was achieved in Pseudomonas putida cells. The recombinant protein had biological activity.