Tri Suciati

Codon optimization for high level expression of human bone morphogenetic protein – 2 in Escherichia coli

Codons in the open reading frame (ORF) encoding for human bone morphogenetic protein-2 (hBMP-2) were optimized to reach high level expression in Escherichia coli. The optimization was done by the computer programs DNA works and DNA Star according to Thermodynamically Balanced Inside Out (TBIO) approach. The ORF consisting of 342 base pairs (bp) was assembled using two-steps Polymerase Chain Reaction, cloned into a pGEM-T vector with a mutation rate of 6.38 bp per kb and transformed into E. coli JM109. After a DNA sequence confirmation, mutation-free ORF was subcloned into pET32b and transformed into E. coli BL21(DE3). The rhBMP-2 was produced as a thioredoxin-his-tag fusion protein at relatively high level, approximately 60% of total intracellular proteins as inclusion bodies (IB), with a yield of 1.39 g per liter culture. Solubilization of IB gave soluble monomer rhBMP-2 with a recovery of 13.6% and refolding of soluble rhBMP-2 produced dimeric forms with a yield of 8.7%. The size and identity of the purified rhBMP-2 was confirmed by nano-LC-MS/MS2 analysis. Our work demonstrates for the first time that by using TBIO approach, a codon-optimized ORF encoding for rhBMP-2 protein can be expressed at high level in E. coli expression system.

Electrospinning of Poly(vinyl alcohol)/Chitosan via Multi-Nozzle Spinneret and Drum Collector

Poly (vinyl alcohol) (PVA)/chitosan nanofibers membranes were successfully prepared by electrospinning employing a multi-nozzle and a drum collector. The use of multi-nozzle and drum collector system is intended to produce stacked nanofibers to form a membrane as a matrix for burn wound healing application. The membranes thickness was controlled by varying applied voltage, flow rate, drum collector rotating speed, and nozzle-collector distance. The electrospinning process and nanofibers formation on the collector was observed using a digital camera. The scaling relationship between current-flow rate and current-voltage for PVA/chitosan fulfills power laws of I ~ Qα and I ~ Eβ, respectively.

Mass Production of Stacked Styrofoam Nanofibers Using a Multinozzle and Drum Collector Electrospinning System

Electrospinning offers the unique ability to produce fibers with very small diameters down to a few tens of nanometers and an attractive mechanical appearance as well as the controllability of morphology, surface and pores structure. However, the main drawback of the conventional electrospinning technique is its low productivity. In order to produce high yield nanofibers, the production rate of nanofibers must be improved. The multinozzle and drum collector electrospinning system was then developed to overcome the problem. The multinozzle sub-system was used to increase the production rate of nanofibers while the drum collector was utilized to maintain the uniformity and thickness of stacked nanofibers. A solution prepared by dissolving waste styrofoam in the mixture of tetrahydrofuran (THF), citronella oil and cajuput oil was the precursor to produce the stacked styrofoam nanofibers.