Expression And Purification Of Midkine From Pichia Pastoris And Its Establishment Of Yeast Seed Bank

Midkine (MK) is one of the heparin-binding growth factors that are stronglyexpressed during embryogenesis. It is vigorously involved in development andregeneration of normal tissues, as well as in inflammation and tumorigenesis. Weestablished a Pichia pastoris-based MK protein expression system, by which wesuccessfully resolved the problems about the protein renaturation and homogeneity.The protein derived from the test animal species is reasonable and necessary for theimmunological reason. Here, a successful method for MK production is described andenough amount of native MK with high quality standard was obtained for the further study.Also,we established a good seed bank after verification.To facilitate further studies of recombinant human MK (rhMK), we developed astrategy for expressing and purifying rhMK using Pichia pastoris expression system. Thisstrategy is simple and efficient with construction of expression vector, high-densityfermentation and one-step of cation ion exchange chromatography. The protein has Highpurity, and proved to be biological active in the UMR-106cells.Furthermore, Theendotoxin level of thefinal formulated rhMK protein solution was1.4-7.0EU/mg protein.Recombinant mouse MK (rmMK) was efficiently expressed under the control of theAOX1promoter in Pichia pastoris, X-33strain line, and secreted into fermentation brothin high-density fermentation. Totally,380mg of rmMK, containing authentic andtruncated forms, was secreted into1.2L of medium, and280mg rmMK was obtained after one-step purification on a50ml SP Sepharose Fast Flow column. The purifiedprotein was characterized and identified to be the mature.The purity was determined to beabove99%with high performance liquid chromatography.The biological activity of thefinal product was verified with migration assay on UMR-106cells. The strategy willfacilitate the physiological and pathological research of MK and have reference values forthe expression and purification of other proteins.Finally, we established the original seed bank, the main seed bank and the workingseed bank after successfully establishing bacteria strains carrying the expression plasmidof rhMK for fermentation. Yeast strains from all the three levels of seed banks showtypical yeast clone morphology on YPD plates, and show typical characteristics of yeastwhen observed under microscope. The rhMK expression level of all the yeast strains areconsistent with each other, and using PCR method, we have confirmed that the exogenousmidkine gene exist in such yeast stains with stability of over95%. Both the indexes showthat the yeast strains we construct for the seed banks absolutely meet the correspondingscientific formulates.