Perpustakaan Digital ITB

Protein nanopar+cles (PNPs) capable of self-assembly such as bacteriophage virus-like par+cles (VLPs) and polymeric ferri+n nanopar+cles may serve as a suitable carrier for vaccines due to the advantages they propose. VLPs are rela+vely thermostable and par+cularly immunogenic – inducing a higher immune response while being non-infec+ous due to the absence of viral gene+c material. Meanwhile, ferri+ns exhibit high thermal as well as pH stability and its unique structure allows for internal and external modifica+ons. Furthermore, their self-assembling capability makes them economical in terms of manufacturing rela+ve to other exis+ng PNP fabrica+on methods as they produce nanocages. This experiment aims to produce VLPs and ferri+n using previously op+mized condi+ons and to the purify them as well as to characterise its purified results. The VLPs were obtained from three varying bacteriophage origins, namely: P, Q, and R while ferri+ns were obtained from three thermophilic bacteria, namely: X, Y, and Z. Overproduc+on of all proteins were done using IPTG as an inducer, exhibi+ng consistent induc+on +me, wet cell weight, and protein profile using SDS-PAGE. Op+mal purifica+on condi+ons for VLP proteins were achieved using affinity chromatography with various imidazole concentra+ons of equilibra+ng, washing, and elu+ng buffers. P and Q VLP proteins were purified in a respec+ve order of buffer concentra+ons 25 mM; 10 mM; and 250 mM imidazole while that of R was 50 mM; 100 mM; and 2000 mM imidazole. A method to op+mize ferri+n purifica+on was established using ferri+n Z by thermal precipita+on at 80°C with ion-exchange chromatography as the ini+al purifica+on steps followed by size-exclusion chromatography as the polishing stage. Purified VLPs were characterised using SDS-PAGE, showing a protein size of 29.04 kDa for proteins P and Q as well as 30.25 kDa for protein R. Purified Z ferri+n was characterised with SDS-PAGE and dynamic light scagering (DLS), exhibi+ng a narrow-peaked species with a radius of 13.3 nm with a size of approximately 19kDa.