No abstract

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence.

Recombinant human interferon alpha-2a (hIFNα-2a) is therapeutic protein that widely used in hepatitis B/C and several cancer treatments. We developed higher molecular weight of hIFNα-2a to improve protein pharmacokinetic profile. The protein was designed as a fusion protein with human serum albumin as protein tag. The protein was produced in Pichia pastoris with 85 kDa in size. This research was aimed to purify, characterize and determine the stability expression of open reading frame (ORF) encoding Fusion and Non fusion forms of hIFNα-2a. The proteins were purified using affinity chromatography and characterized using SDS PAGE and Western Blotting methods. Protein recovery yield was determined by ELISA. Stability expression was applied in generation time until 90th generation. The results showed that the Fusion and Non fusion proteins were successfully purified with 74-79% of protein recovery. The proteins can be recognized by specific monoclonal antibody and verified as hIFNα-2a Fusion and Non fusion with 85 kDa and 19 kDa in size respectively. The expression stability showed that the proteins were still produced in Pichia pastoris until 90th generation time with no significant difference of expression level. To conclude, the expression level of ORFs encoding Fusion and Non fusion hIFNα-2a was stable until 90th generations.

Nanoparticles have been promptly studied and developed for oral protein delivery. Selection of excipients and formulation method depends on the physicochemical characteristics of the protein carried. Therefore, this study aims to design a formulation and characterize the alginate coated chitosan nanoparticles which carried different protein. Alginate coated chitosan nanoparticles were formed using an ionic gelation method. Optimization was done by varying the parameter such as crosslinker concentration, agitation method, rate, and time.  The results show that chitosan nanoparticles formed by sonication using sodium tripolyphosphate (STPP): chitosan (1:0.8) was the best method to form nanoparticles. The particle size and polydispersity index (PDI) of bovine serum albumin (BSA) and lysozyme nanoparticles were 812.2 nm and 0.412 for BSA while 793.3 nm and 0.438 for lysozyme. Encapsulation efficiency (EE) of BSA is 52 % and lysozyme is 68 %. In vitro tests on acidic/gastric conditions showed that lysozyme (±32 %) was released faster than BSA (±10 %) during the 24 h incubation. Under neutral/terminal intestine conditions, percentage of BSA (±17 %) release is slightly higher than lysozyme (±13 %) for 8 h incubation period. It was concluded that the formulation of alginate coated chitosan nanoparticles in this study appears to be more effective for BSA delivery than lysozyme. HIGHLIGHTS Nanoparticle vesicular system is an effective approach for protein drug delivery system Factors affecting the formation of chitosan nanoparticle are crosslinker concentration, agitation method, rate, and time Alginate coated chitosan nanoparticle was more effective for oral delivery of BSA compared to lysozyme GRAPHICAL ABSTRACT

No abstract