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SOETIJOSO SOEMITRO

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Author-ID : 433766

Biochemistry, Genetics & Molecular Biology Computer Science & IT Immunology & microbiology Medicine & Pharmacology

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Codon Optimization and Chaperone Assisted Solubilization of Recombinant Human Prethrombin-2 Expressed in Escherichia coli SARONOM SILABAN; IMAN PERMANA MAKSUM; SHABARNI GHAFFAR; KHOMAINI HASAN; SUTARYA ENUS; TOTO SUBROTO; SOETIJOSO SOEMITRO
Microbiology Indonesia Vol. 8 No. 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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.

Heterologous Expression of Î±-Amylase from Saccharomycopsis fibuligera R64 and its Tyr401Trp Mutant in Pichia pastoris RIEZKI AMALIA; WANGSA TIRTA ISMAYA; FERNITA PUSPASARI; KHOMAINI HASAN; TOTO SUBROTO; DESSY NATALIA; SOETIJOSO SOEMITRO
Microbiology Indonesia Vol. 10 No. 1 (2016): March 2016
Publisher : Indonesian Society for microbiology

Î±-Amylase from Saccharomycopsis fibuligera R64 is a non-adsorbing raw-starch degrading enzyme, a unique characteristic. This character is difficult to explain in the absence of its three-dimensional structure. Here we discuss the expression of a-amylase from Saccharomycopsis fibuligera in Pichia pastoris and the effect of site directed mutagenesis on its activity. A model based on the structure of its homologs suggested mutation of codon of Tyr401 into that of a Trp residue. An activity study using whole cells P. pastoris showed similar substrate degradation rates by cells carrying either the native or mutant amylase encoding gene. However, the purified enzyme of the mutant strain showed faster starch hydrolysis.

Cloning, Expression, and Functional Characterization of Autoactivated Human Prethrombin-2 Synthetic Gene by Using Pichia pastoris SMD1168 As a Host TOTO SUBROTO; WULAN PERTIWI; MUHAMMAD FADHILLAH; KHOMAINI HASAN; OGI BUDIANTORO; SUTARYA ENUS; SOETIJOSO SOEMITRO
Microbiology Indonesia Vol. 10 No. 2 (2016): June 2016
Publisher : Indonesian Society for microbiology

Prethrombin-2 is a thrombin precursor that has important role in blood coagulation. It is the smallest precursor which is activated into thrombin by FXa prior to coagulation process. However, as a commercial theurapetic protein in fibrin sealant component, prethrombin-2 must be activated by ecarin before used. Thus, the production process of this protein needs further purification. In order to eliminate ecarin activation step and to increase production efficiency, we designed, cloned and expressed the recombinant autoactivated human prethrombin-2 in Pichia pastoris SMD1168. The variant was designed with 4 mutations, E40A, D47A, G48P, and E52A, following the result of a previous study. The synthetic variant gene was first optimized to conform with P. pastoris codon preference. The optimized synthetic gene was cloned in pD912 plasmid using XhoI and SacII restriction enzymes. The transformed P. pastoris was selected on agar plate supplemented with 1,000 Âµg.mL-1 Zeocin as a selection marker. This study showed that autoactivated prethrombin-2 was succesfully expressed extracellularly by P. pastoris SMD1168. The activity of recombinant autoactivated prethrombin-2 using a chromogenic substrate S-2238 was 0.540 unit/mg. Taken together, these results demonstrated that autoactivated human prethrombin-2 was successfully produced extracellularly in P. pastoris.

Co-Authors DESSY NATALIA FERNITA PUSPASARI IMAN PERMANA MAKSUM KHOMAINI HASAN Muhammad Fadhil Z, Rusdi Noor Rosa (p:43-50) OGI BUDIANTORO Purba Upay Riezki Amalia SARONOM SILABAN Shabarni Gaffar SHABARNI GHAFFAR SUTARYA ENUS Toto Subroto WANGSA TIRTA ISMAYA Wulan Pertiwi

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Journal : Microbiology Indonesia