Auto-activated Thrombin Expression from Pichia pastorisSMD1168 Optimized by Plackett-Burman Design and Response Surface Methodology
Journal Title: IOSR Journal of Pharmacy (IOSRPHR) - Year 2017, Vol 7, Issue 9
Abstract
Abstract: Recombinant thrombin (rThrombin) has been used topically as one component of fibrin sealant as the tissue adhesive. Recombinant prethrombin-2 is used as the precursor to generate rThrombin which is then activated by ecarin. Autoactivated thrombin (autoTrm) can be produced by altering four amino acids (EDGE) in prethrombin-2 thus obviate the use of ecarin and simplify the purification process. In this study, Pichia pastorisSMD1168 was used to producing autoTrm in flask culture. Influencing factors on P. pastorisSMD1168 culture in producing autoTRM were screened by Plackett-Burman (PB) design and the optimization of was carried out by Response Surface Methodology. The medium used for cultivation was BMGH for growth phase and Pichia Trace Minerals (PTM1) supplemented Basal Salt Medium (BSM) for induction phase. The influencing factors (PTM1 concentration, methanol concentration, induction temperature,induction time, and pH) on autoTrmexpression werescreenedby PB design then were optimized by Response Surface Methodology (RSM) with Central Composite Design.It was revealed that PTM1 concentration (%) and pH had significant influence (P < 0.05) on autoTrm expression level based on PB design. The optimum%PTM1 and pH revealed by (RSM) was 2.05% and 4.2, respectively
Authors and Affiliations
Muhammad Fadhlillah, Wulan Pertiwi, Ersanda Hafiz, Sheila Chairunnisa, Eria S. Widyaningsih, Inten Ihsannunisa, Toto Subroto
Keywords
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