The α-mating factor secretion signals and endogenous signal peptides for recombinant protein secretion in Komagataella phaffii.

BACKGROUND: The budding yeast Komagataella phaffii (Pichia pastoris) is widely employed to secrete proteins of academic and industrial interest. For secretory proteins, signal peptides are the sorting signal to direct proteins from cytosol to extracellular matrix, and their secretion efficiency directly impacts the yields of the targeted proteins in fermentation broth. Although the α-mating factor (MF) secretion signal from S. cerevisiae, the most common and widely used signal sequence for protein secretion, works in most cases, limitation exists as some proteins cannot be secreted efficiently. As the optimal choice of secretion signals is often protein specific, more secretion signals need to be developed to augment protein expression levels in K. phaffii. RESULTS: In this study, the secretion efficiency of 40 α-MF secretion signals from various yeast species and 32 endogenous signal peptides from K. phaffii were investigated using enhanced green fluorescent protein (EGFP) as the model protein. All of the evaluated α-MF secretion signals successfully directed EGFP secretion except for the secretion signals of the yeast D. hansenii CBS767 and H. opuntiae. The secretion efficiency of α-MF secretion signal from Wickerhamomyces ciferrii was higher than that from S. cerevisiae. 24 out of 32 endogenous signal peptides successfully mediated EGFP secretion. The signal peptides of chr3_1145 and FragB_0048 had similar efficiency to S. cerevisiae α-MF secretion signal for EGFP secretion and expression. CONCLUSIONS: The screened α-MF secretion signals and endogenous signal peptides in this study confer an abundance of signal peptide selection for efficient secretion and expression of heterologous proteins in K. phaffii.

Can More Nanoparticles Induce Larger Viscosities of Nanoparticle-Enhanced Wormlike Micellar System (NEWMS)?

There have been many reports about the thickening ability of nanoparticles on the wormlike micelles in the recent years. Through the addition of nanoparticles, the viscosity of wormlike micelles can be increased. There still exists a doubt: can viscosity be increased further by adding more nanoparticles? To answer this issue, in this work, the effects of silica nanoparticles and temperature on the nanoparticles-enhanced wormlike micellar system (NEWMS) were studied. The typical wormlike micelles (wormlike micelles) are prepared by 50 mM cetyltrimethyl ammonium bromide (CTAB) and 60 mM sodium salicylate (NaSal). The rheological results show the increase of viscoelasticity in NEWMS by adding nanoparticles, with the increase of zero-shear viscosity and relaxation time. However, with the further increase of nanoparticles, an interesting phenomenon appears. The zero-shear viscosity and relaxation time reach the maximum and begin to decrease. The results show a slight increasing trend for the contour length of wormlike micelles by adding nanoparticles, while no obvious effect on the entanglement and mesh size. In addition, with the increase of temperature, remarkable reduction of contour length and relaxation time can be observed from the calculation. NEWMS constantly retain better viscoelasticity compared with conventional wormlike micelles without silica nanoparticles. According to the Arrhenius equation, the activation energy Ea shows the same increase trend of NEWMS. Finally, a mechanism is proposed to explain this interesting phenomenon.

Investigation of Novel Triple-Responsive Wormlike Micelles.

Smart wormlike micelles with stimuli-tunable rheological properties may be useful in a variety of applications, such as in molecular devices and sensors. The formation of triplestimuli-responsive systems so far has been a challenging and important issue. In this work, a novel triplestimuli (photo-, pH-, and thermoresponsive) wormlike micelle is constructed with N-cetyl-N-methylmorpholinium bromide and trans-cinnamic acid (CA). The corresponding multiresponsive behaviors of wormlike micellar system were revealed using cryogenic transmission electron microscopy, a rheometer, and 1H NMR. The rheological properties of wormlike micellar system under different temperatures, pH conditions, and UV irradiation times are measured. As confirmed by 1H NMR, chemical structure of a CA molecule can be altered by the multiple stimulation from an exotic environment. We expect it to be a good model for triple-responsive wormlike micelles, which is helpful to understand the mechanism of triple-responsiveness and widen their applications.

Design and Study of a Novel Thermal-Resistant and Shear-Stable Amphoteric Polyacrylamide in High-Salinity Solution.

Abstract: Water-soluble polymers are widely used in oilfields. The rheological behaviors of these polymers in high-salinity solution are very important for stimulation of high-salinity reservoirs. In this work, a novel thermal-resistant and shear-stable amphoteric polyacrylamide (PASD), prepared from acrylamide (AM), sodium styrene sulfonate (SSS), and acryloxyethyl trimethylammonium chloride (DAC) monomers, was prepared by free-radical polymerization in high-salinity solution. The amphoteric polyacrylamide was characterized by Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance spectroscopy (¹H NMR), elemental analysis, thermogravimetric analysis (TG), and scanning electron microscopy (SEM). The amphoteric polyacrylamide exhibited excellent salinity tolerance. The slow increase in apparent viscosity of the polymer with increase in salinity was interesting. The amphoteric polyacrylamide showed perfect temperature resistance in high-salinity solution. The viscosity retention reached 38.9% at 120 °C and was restored to 87.8% of its initial viscosity when temperature was decreased to room temperature. The retention ratio of apparent viscosity reached 49.7% at 170 s-1 and could still retain it at 25.8% at 1000 s-1. All these results demonstrated that PASD had excellent thermal-resistance and shear-stability in high-salinity solution. We expect that this work could provide a new strategy to design polymers with excellent salinity-tolerance, thermal-resistance, and shear-stability performances.