Pemurnian Pretrombin-2 Manusia Rekombinan di Escherichia coli untuk Produksi Trombin sebagai Komponen Lem Fibrin

Abstract

Abstrak: Pretrombin-2 (PT2) adalah precursor trombin yang berperan mengubah fibrinogen menjadi fibrin untuk proses penutupan luka. Bahan ini dapat diterapkan sebagai pengganti teknik jahitan bedah mata. Sistem pemurnian yang dimediasi intein untuk memurnikan protein menarik untuk dikembangkan, karena protein diperoleh dengan satu tahap pemurnian, mampu melakukan pemotongan sendiri (self-splicing), protein dapat difusikan pada (pemotongan PT2 dari penanda intein terinduksi pH dan suhu) dan pada C-terminal (pemotongan PT2 dari penanda intein terinduksi pereaksi tiol). Dalam studi ini, kami memurnikan fusi PT2 hasil ekspresi E. coli BL21(DE3) ArcticExpress dalam kolom matriks kitin yang dimediasi intein. PT2 difusikan dengan suatu tag pada posisi Nterminalnya, yang mengandung urutan pengode intein SspDnaB diikuti oleh domain pengikat kitin (CBD). Selanjutnya fusi PT2 diekspresikan pada inang E. coli, selanjutnya dimurnikan dalam kolom matriks kitin. Proses pemotongan PT2 dari penanda intein diinduksi perubahan pH dan suhu dalam kolom.Fusi PT2 berhasil dimurnikan dalam matriks kitin yang dimediasi intein. Pemotongan fusi PT2 dari penanda intein terinduksi buffer pH 6,5 dan suhu inkubasi 25 o C selama 48 jam.

Expression of SARS-CoV-2 Nucleocapsid (N) Recombinant Protein Using Escherichia coli System

“…A high GC value (>70%) can reduce efficiency and inhibit translation process, while a low GC value (<30%) can causes a delay in the transcriptional elongation process (Rosano and Ceccarelli 2009). According to (Gustafson et al 2014;Merkl 2003;Silaban and Subroto 2016), the differences in the percentage of GC content between the target gene and host expression causes codon bias, where a high codon bias can reduce expression levels and was thought to cause translation errors and produce inactive complex aggregate proteins that cause degradation into different fragments. The codon optimization of SARS-CoV-2 N synthetic gene increased the CAI value to 0.96, theoretically the CAI value >0.8 was predicted to be suitable for expression in E. coli hosts (Carbone et al 2003;Xia 2007).…”

Temperature effect on expression of recombinant human prethrombin-2 in Escherichia coli BL21(DE3) ArcticExpress

“…This system remains to be one of the simplest and most preferred bacterial expression systems for heterologous expression of proteins for academic researches as well as of industrial and therapeutic recombinant proteins (Chen, 2012;Osadska et al 2014). However, inability of E. coli to express proteins with high molecular weight, disulphide bond rich, or posttranslational modification become a bottleneck.Prethrombin-2 had previously been expressed in E. coli (Choi et al 1989;So et al 1992;DiBella et al 1995;Soejima et al 2001;Silaban et al 2015;;SIlaban et al 2017). Soejima et al (2001), reported a new method of easy expression and efficient refolding of recombinant human prethrombin-2 using an E. coli expression system, which could be usable also for the pharmaceutical industry.…”

“…The final yield of purified prethrombin-2 was 0.5-1%, which means that from 1 liter of E. coli bacterial culture only 1 mg of the recombinant protein could be recovered. Prethrombin-2 is expressed in E. coli, only a small amount of thrombin is active due to the formation of inclusion bodies (Freydell et al 2007;Silaban et al 2016). Inclusion bodies is a dynamic structure that formed by an imbalance between the aggregates and soluble proteins from E. coli.…”