J.H Choi

Secretory and extracellular production of recombinant proteins using Escherichia coli

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.

Optical Outcoupling Enhancement in Organic Light‐Emitting Diodes: Highly Conductive Polymer as a Low‐Index Layer on Microstructured ITO Electrodes

Organic light-emitting diodes (OLEDs) are now entering mainstream display markets and are also being explored for next-generation lighting applications. In both types of applications, high external quantum efficiency (EQE) is of premium importance for both low power consumption and long lifetime. It is well known that one of the bottlenecks in achieving high EQE in OLEDs is the low light-extraction efficiency, which is limited to <20%, mostly because total internal reflections occurring at interfaces between optically distinctive layers confine a significant portion of the light within the substrate (1⁄4 ‘‘substrate-confined’’ mode) or within the organic/indium tin oxide (ITO) layers (1⁄4 ‘‘wave-guided’’ mode). Hence, many device structures have been proposed to extract light that would not normally be outcoupled: some have attempted to extract the light that is confined in a substrate by introducing structures such as microlens array (MLA) or pyramidal arrays on the backside of the substrate, where other research groups have tried to extract the light that is confined within organic/ITO layers by introducing optical structures such as photonic crystals or low-index grids that can disrupt the wave-guiding of the light within the organic/ITO layers. The lattermay be carried out in a direct way by converting wave-guided modes directly into outcoupled modes or in an indirect way by converting wave-guided modes into substrate-confined modes and then extracting them with structures mentioned in the former approach. Criteria for choosing a specific method or structure over others depend on the target applications: for display applications, methodologies such as MLA and substrate structuring are often avoided due to their optical blurring effect; for lighting applications, such methods are readily accepted, but complex processes that add too much cost are generally not welcomed. In both cases, compatibility with a common fabrication technique and large-area fabrication is strongly preferred, and the Lambertian angular dependence and the absence of spectral dependence are also preferred in most situations. Here we introduce a novel anode structure based on micropatterned ITOs coated with high-conductivity (HC)-grade poly(3,4ethylenedioxythiophene): poly(styrene sulfonate) (PEDOT:PSS) layers. This proposed electrode structure can improve the outcoupling efficiency of OLEDs in a relatively simple way without severe spectral dependence, blurring (optional), or deviation from the normal angular dependence. Figure 1 illustrates a tilted top-view and cross-section of the proposed anode structure and its working principle. ITO layers are patterned so that the square opening (Wo Wo) repeats in a square lattice layout with a spatial period ofWt. For simplicity, we consider a situation where Wt1⁄4 2 Wo. In this case, the width of ITO strips (WITO (1⁄4Wt–Wo)) next to each opening equalsWo, and the ITO-less portion is 25% per each unit cell. The spatial period and the dimension of openings are chosen to be sufficiently larger than the emission wavelength so that a geometric optic approach can be valid and spectral dependence may be ignored. Each pattern may have a taper angle utaper, as defined in Figure 1a. A high-conductivity PEDOT:PSS (Baytron PH 500, HC Starck, Inc.) layer is coated throughout the anode area over the patterned anode. Organic layers and metal cathodes are then deposited to complete the device. Note that the light emitted with a small angle within the emission layer, which would normally be wave-guided throughout the organic/ ITO layers, is now guided either solely within organic layers (the ray in red coming from the left side in Figure 1b) or solely within ITO layers (the ray in blue coming from the right side in Figure 1b), because the refractive index ( 1.42 at l1⁄4 550 nm) of PEDOT:PSS is lower than those of ITOs and typical organic layers used in OLEDs. Upon hitting the structured region once or multiple times, the guided light will change its direction so that it can be directly coupled out. Some portion of the wave-guided mode can also be converted to a substrate-confined mode (see Figure S1 in Supporting Information). It has to be noted that patterned ITO electrodes alone without the PEDOT:PSS overcoat would not work effectively, because organic layers and ITO layers are optically almost identical due to their similar refractive indices. The low refractive index of a PEDOT:PSS layer is indeed the characteristic that enables internal reflections at the structured interfaces, which are among the key processes that must occur in order for the guided light to be converted to an outcoupled or a substrate-confined mode. In addition to the optical benefits noted above, it is also critical that, in the region without ITO, the HC-grade PEDOT:PSS layer provides an electrical sheet conduction and works as an anode independently so that there is no inactive area in the devices. In fact, we note each anode region consisting solely of PEDOT:PSS is surrounded by ITO electrodes, resembling the conductive grid structure suggested by Leo and his coworkers that can ensure

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence.

Abstract New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation.

Highly transparent organic light-emitting diodes with a metallic top electrode: the dual role of a Cs 2 CO 3 layer

Highly transparent organic light-emitting diodes (TrOLEDs) are demonstrated using damage-free top cathodes of Cs2CO3/ Ag capped with ZnS layers. The presence of ultrathin Cs2CO3 layers not only improves the electron injection properties but also makes Ag thin films more continuous and uniform, resulting in ideal top electrodes with low sheet resistance and high transmittance. The combination of the uniform Ag morphology enabled by Cs2CO3 and the optimized thickness of ZnS capping layers results in TrOLEDs that have a peak transmittance as large as 80% with a luminous transmittance of 76.4%. These TrOLEDs exhibit a low turn-on voltage of 2.6V due to injection improvement by the Cs2CO3 layers.

Economic considerations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterial fermentation.

Abstract The process for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)] by bacterial fermentation and its recovery was analysed. The effects of various factors such as P(3HB/V) content, P(3HB/V) productivity, P(3HB/V) yield and 3-hydroxyvalerate (3HV) fraction in P(3HB/V) on the production cost of P(3HB/V) were examined. The increase in the 3HV yield on a carbon source did not significantly decrease the production cost when the 3HV fraction was 10 mol%, because the cost of the carbon substrate for 3HV was relatively small in terms of the total cost. However, at a 3HV fraction of 30 mol%, the 3HV yield on a carbon source had a significant effect on the total P(3HB/V) production cost. The production cost of P(3HB/V) increased linearly with the increase in the 3HV fraction in P(3HB/V).

Effect of nutrient deficiency on accumulation and relative molecular weight of poly-β-hydroxybutyric acid by methylotrophic bacterium, Pseudomonas 135

SummaryPseudomonas 135, a facultative methylotroph, was cultivated on methanol as a sole carbon and energy source for the accumulation of poly-β-hydroxybutyric acid (PHB). The cells grew fairly well on minimal synthetic medium containing 0.5% (v/v) of methanol at pH 7.0 and 30° C. The maximum specific growth rate was determined to be 0.26–0.28 h−1 with a growth yield of 0.38 in the optimized growth medium. For stimulation of PHB accumulation in the cells, deficiency of nutrients such as NHinf4sup+, Mg2+ and POinf4sup3−was crucial even though cell growth was significantly suppressed. The PHB content of a 40-h culture was determined to be 37% of the total cell mass in NHinf4sup+-limited medium, 42.5% on Mg2+-deficient medium, and 34.5% on POinf4sup3−-deficient medium. The maximum content of PHB in the cells could reach 55% in NHinf4sup+-limited fed-batch culture. The average relative molecular eight determined by gel permeation chromatography was 3.7 × 105 in NHinf4sup+-limited culture, 2.5 × 105 in Mg2+-deficientmedium, and 3.1 × 105 in POinf4sup3−-deficient medium. Polydispersity determined in each culture was relatively high (about 10–11). The solid PHB had a melting temperature of 173° C.

Enhanced light extraction in organic light-emitting devices: Using conductive low-index layers and micropatterned indium tin oxide electrodes with optimal taper angle

We present our study on organic light-emitting diodes (OLEDs) in which outcoupling is enhanced based on a bilayer electrode consisting of a conductive low-index layer and micro-patterned indium tin oxide (ITO) layers. Optical simulation reveals that the taper angle of an ITO pattern is among the most critical parameters influencing the outcoupling efficiency in the proposed structure. A fabrication method based on a lift-off process is then employed to control the taper angle of the ITO pattern to be in the optimal range. OLEDs with the proposed electrode structure exhibit 50%–70% enhancement in external quantum efficiency over reference devices.

Digital-Mode Organic Vapor-Jet Printing (D-OVJP): Advanced Jet-on-Demand Control of Organic Thin-Film Deposition

Digital-mode organic vapor-jet printing (D-OVJP) is demonstrated by producing a series of organic vapor jets. D-OVJP not only inherits all the benefits of a conventional OVJP but also provides an advanced, straightforward control over organic deposition with a pixel-to-pixel precision. Digitally-controlled film thickness and high-performance thin-film transistors are demonstrated with D-OVJP, proving its potential applicability to organic electronics and related areas.