Pustaka Ilmiah Universitas Padjadjaran

Abstrak

Codon Optimization and Chaperone Assisted Solubilization of Recombinant Human Prethrombin-2 Expressed in Escherichia coli

Pretrombin-2 (PT2) adalah prekursor trombin yang berperan dalam mengubah fibrinogen menjadi fibrin selama proses pembekuan darah. Penelitian sebelumnya melaporkan bahwa ekspresi pretrombin-2 manusia rekombinan (rhPT2) dalam inang Escherichia coli selalu menghasilkan badan inklusi. Tujuan penelitian ini adalah mencari strategi dalam mengekspresikan rhPT2 dalam bentuk terlarut dalam inangE. coli. Desain eksperimen meliputi perancangan dan optimasi kodon pretrombin-2 manusia (hpt2) serta mengoptimasikan ekspresi rhPT2 terlarutdengan menggunakan 4 galurE. coli. Gen hpt2 menunjukkan codon adaptation index (CAI) sebesar 0.336 dengan presentase GC 56.8%, sementara hasil optimasi menunjukkan CAI sebesar 1.000 dengan presentase GC sebesar 53.1%. Selanjutnya, gen hpt2 berhasil diklon ke dalam vektor ekspresi pTWIN1.Uji ekspresi protein rhPT2 terhadap 4 galur inang E. coli menunjukkan bahwa galur ArcticExpress mampu mengekspresikan protein rekombinan dalam bentuk terlarut walapun sejumlah protein juga terekspresi dalam bentuk badan inklusi. Sementara galur lain menunjukkan hasil ekspresi protein rekombinan selalu dalam bentuk badan inklusi. Selain preferensi kodon hpt2 yang sesuai dengan E. coli, peran chaperon Cpn60/Cpn10 yang terdapat pada E. coli ArcticExpress diprediksi memiliki peran yang sangat krusial dalam membantu pelipatan rhPT2, sehingga dapat diekspresikan dalam bentuk terlarut.

Prethrombin-2 (PT2) is a thrombin precursor, which plays a role in the conversion of fibrinogen into fibrin during blood clotting process. Previous study reported that the expression of human prothrombin-2 (rhPT2) in Escherichia coli formed inclusion bodies. The aim of this study was to establish a strategy to express a soluble rhPT2 in E. coli. This study was animed to design and codon optimize human prethrombin-2 gene as well as to optimize the expression condition using four strains of E. coli. The codon adaptation index (CAI) of the unoptimized hpt2 gene was 0.336, with 56.8% GC content. After optimization, the CAI of optimized hpt2 became 1.000 with 53.1% GC content. The optimized gene was successfully cloned into pTWIN1 expression vector. Expression analysis indicated that only E. coli ArcticExpress strain could successfully express a soluble recombinant rhPT2 protein, with only part of rhPT2 being expressed in insoluble form. However, the rest of the E. coli strains used in the experiments failed to express the rhPT2 in soluble form. We are deducing that the success in achieving soluble expression was not only due to the availability of chaperonins Cpn60/Cpn10, which played a crucial role in the protein folding in E. coli ArcticExpress strain, but also due to the codon optimization of hpt2 gene.