DEVELOPMENT OF BIOSIMILAR RECOMBINANT HUMAN INTERFERON-?2B THROUGH PERIPLASM EXPRESSION

• Main Author: Jessica, Adhitya
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45230
• Institution: Institut Teknologi Bandung

Human recombinant interferon alfa2b (rhIFN?2b) as a biosimilar product must have an identic amino acid sequence to the native form. Unauthenticity of N-terminal amino acid can be overcomed by peripasmic expression. Construction of recombinant plasmid genes to transformation into E. coli TOP10 had been done by previous researchers ( Retnoningrum et al., 2012). This study focused on the optimization of overproduction, periplasmic protein isolation and purification process. Isolation of periplasmic proteins has been developed by several modification osmotic shock. rhIFN?2b was purified by anionic exchange chromatography with gradient of pH and NaCl concentration. Then, gel filtration chromatography also was applied in the purification process. rhIFN?2b was characterized with Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE), and Western blot. The condition for overproduction of rhIFN?2b in the periplasm region are 0.1 mM IPTG as inducer, in 25 o C, and for 18 hours. Osmotic shock seems to be promising technique among other techniques, especially for large-scale operation. Analysis of cytoplasm crude extract with SDS PAGE showed thick band above 18,4 kilo Dalton. This band was parallel with thin band shown at SDS PAGE profile of periplasm crude extract. Analysis of cytoplasm and periplasm extract with Western blot showed purple band above 18,4 kilo Dalton also.Overproduction condition and osmotic shock methods as optimization results can be used to obtain the periplasmic fraction. It requires LC MS / MS analysis to determine whether rhIFN?2b was still in a state of fusion with enterotoksi II. It also requires further purification optimization in order to produce high amounts of protein in the periplasm.