[Design of the human GM-CSF gene using the polymerase chain reaction and its expression in Pseudomonas putida cells]

Construction of human GM-CSF gene was conducted by the PCR technique. Four exons of GM-CSF gene were synthesized on the basis of human blood DNA using thermostable Tth DNA polymerase. Synthetic oligonucleotides were used as primers. The oligonucleotides contained sequences complementary to the ends of exons. Joining of exons was conducted by reciprocal complementation of the terminal sequences, followed by filling and amplification of the joined products. In most cases the effective synthesis of exons and joined products was possible only after optimizing the polymerase reaction conditions for each primer pair. Determination of the nucleotide sequence of the synthetic gene showed complete identity with the natural one. The gene was introduced into an expression vector under control of the promoter tandem (tac+lac). Expression of the GM-CSF gene was obtained in Pseudomonas putida cells. The recombinant protein had biological activity.