Affinity Purification Of Egg Yolk Immunoglobulins Using PM Protein

Egg yolk immunoglobulin(IgY)is a superior functional equivalent to mammalian Ig G in many applications.However,as a drawback of IgY application,it has been difficult to acquire refined,highly-purified IgY due to some unique biological characteristics of IgY.Protein pM coding sequence(NC 000908.2)was amplified by polymerase chain reaction(PCR).The PCR products were analyzed on a 12% agarose gel.Protein pM was expressed by E.coli BL21/DE3 cells in soluble under 0.4m M IPTG at 20°C with the p ET-32a(+)vector carrying a C-terminal His-tag.C-terminal His-tagged protein pM was purified using His Trap FF Crude with ?KTAxpress purifier.Pull-down assay showed protein pM could capture chicken IgY,duck IgY and duck IgY?Fc,a unique IgY form lacking of CH3 and CH4 and not existed in chicken.Furthermore,the absorbents of IgY hydrolysate(by papain digestion)showed protein pM can bind with IgY-Fab instead of IgY-Fc.Moreover,Western blot assay showed protein pM could bind with IgY-sc Fv.The affinity of protein pM and IgY-sc Fv was 1.922E-10 M detected by surface plasmon resonance(SPR).These results confirmed protein pM can bind with chicken IgY,as well as IgY from Anatidae birds,by blocking the variable region of IgY.Finally,we recovered high-purity IgY from the egg yolk dilution in one-step at p H 3.2 through constructed a protein pM immobilized affinity chromatography column.Gray value analysis confirmed the purity reached 98.7%.The immunoreactivity of the purified IgY was examined by ELISA.A 125 times increase of the concentration of specific IgY was detected compared to the IgY concentration extracted by polyethylene glycol precipitation.Hence,protein pM could be regard as an affinity reagent for the purification of IgY as well as variable region-based antibodies and fragments.