Production process of recombinant placental growth factor

A process for extracting and purifying recombinant Placental Growth Factor (PLGF) protein in dimeric and multimeric active form containing no more than 1.5% of monomeric form, by expression of the PLGF protein in E. Coli cells modified with an inducible expression system comprising an insert coding for the PLGF protein, the process comprising in the following order:

• I) inoculating in a culture medium the E. Coli cells and fermenting the E. Coli cells until obtaining optical density of the culture medium from 14 to 50 at 600 nm (OD600 14-50);
• II) inducing the expression of PLGF protein via the inducible expression system;
• III) extracting the inclusion bodies by lysing the cultured cells, rupturing DNA of the cultured cells, isolating and purifying the inclusion bodies;
• IV) solubilizing the inclusion bodies into a denaturing buffer to form a solution and renaturating the expressed protein PLGF by adding to the solution oxidizing/reducing agents to bring the expressed protein to a dimeric form, at least partially;
• V) passing the solubilized protein on a high flow resin for anion exchange chromatography separating and isolating the dimeric and multimeric forms of the expressed protein from the monomeric form;
• and
• VI) further separating and finally isolating the dimeric and multimeric forms of the expressed protein from the monomeric form by passing them through a reverse-phase chromatography resin with two subsequent elution stages with increasing gradient of an organic solvent separated by an elution stage under isochratic conditions, wherein the organic solvent comprises ethanol, methanol, or acetonitrile, and wherein the monomeric form comprises no more than 1.5% of the total PLGF protein.