Meleshko R. Production of human biologically active prolactin by using baculovirus expresson system

The thesis is devoted to the elaboration of expression of human prolactin by using baculovirus expresson system with following purification of biologically active recombinant hormone. The effective expression of human recombinant prolactin has been reached by using of baculovirus expression system on the basis of: Malacosoma neustria nuclear polyhedrosis virus (NPV) - Antheraea pernyi insect monolayer cell culture and Autographa californica NPV - HF, Sf9 and Sf21 insect monolayer cell culture. Recombinant АcMNPV - Hf cells system demonstrated the highest level of prolactin expression (245 mg/L), due to this fact it was chosen for further work on the way to production of different forms of recombinant prolactin. The HF cells provided the high stable levels of expression of both intracellular (HisPRL) and secreted (MelPRL) prolactin. Combination of conditions, which provide the maximum yield of the recombinant product were established: HF cell suspension cultivation, infecting them with the MOI 1, harvesting prolactin containing cells and medium after 48 hours post infection. Under conditions used recombinant prolactin expression levels were estimated as 500 mg/L and 20-30 mg/L for intracellular and secreted prolactin forms respectively. The recombinant protein purification methods were suggested. Two purification methods for intracellular protein were used: purification using metall-affine chromatography under denaturing conditions with the subsequent renaturing of recombinant protein; prolactin denaturing with the subsequent renaturing and purification of renatured protein using metall-affine chromatography under the native conditions. The heparin-affinity chromatography method was used for purification of secreted prolactin. 95% of purity of recombinant protein was reached, the quantity of the purified product was estimated as 60 mg of the intracellular protein per 1 L of medium and 10 mg of the secreted protein per 1 L of medium. It was shown that approximately 50% of secreted prolactin was glycosylated. Based on the performed research the method of expression and purification of biologically active human prolactin was suggested.