Increased expression of recombinant chitosanase by co-expression of Hac1p in the yeast Pichia pastoris.

Background: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production therein has focused on up-regulating the unfolded protein response (UPR). Objective: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on expression of recombinant chitosanase (rCBS) in P. pastoris. Method: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA and the resulted pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastoris HAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into pGAPZB and then inserted with HIS4 gene from pAO815 to construct the vector pGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and pGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDS-PAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. Results: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulted from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with co-expression of Hac1p or not. Finally, Hac1p co-expression with PAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. Conclusion: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving production of rCBS therein.

Effect of montmorillonite on the oxidative stability of polyphenylene sulfide fibers prepared by melt spinning

Masterbatches of polyphenylene sulfide (PPS)/organic montmorillonite (MMT) composites were produced via melt blending. A self-made spinning equipment was then used to produce the PPS/organic MMT composite fibers by melt spinning directly from the masterbatches. X-ray diffractometer and transmission electron microscope were used to examine the dispersibility of organic MMT. The morphology, tensile property, crystallization behavior, and oxidative stability of PPS fibers were investigated. The results indicated that organic MMT could be uniformly distributed in the PPS matrix to form a mixed dispersion of intercalated and exfoliated structure and influence the longitudinal surface morphology of fibers to become rough. The roughness of composite fibers surface was proportional to the content of organic MMT. The organic MMT nanolayers could act as the heterogeneous nucleating agents to improve the crystallization, and the crystallity of composite fibers increased with the increase of organic MMT content. The breaking strength of composite fibers first increased and then decreased by increasing the amount of organic MMT. After the oxidation treatment, the breaking strength of neat PPS fibers and composite fibers declined, but the degree of breaking strength loss for composite fibers is lower than that of neat PPS fibers. The dynamic oxidation induction temperature of composite fibers also showed a significant increase by adding organic MMT. Moreover, the addition of organic MMT could limit the chemical combination of element sulfur and oxygen, retard the generation of sulfoxide groups, and induce the conversion of sulfur atoms from C-S bond to sulfone for improving oxidative stability.

Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli

Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.

Expression of recombinant mouse cytosolic carboxypeptidase 6 in Escherichia coli

Cytosolic carboxypeptidase 6 (CCP6) is a member of cytosolic carboxypeptidase (CCP) family that catalyze the removal of polyglutamate side chains from protein substrates. Biochemical and biophysical characterization of CCPs requires large quantities of purified proteins. However, no method describing the expression of any mammalian CCP family member from bacteria has been published to our best knowledge. After considerable efforts to improve the solubility of mammalian CCPs expressed in bacteria, including the optimization of induction temperature and by using different receptive cells, we were able to get less expression of mouse CCP6 in soluble fraction of bacterial lysates. We report in this article, the bacterial expression of CCP6 using Arctic Express (DE3) competent cells that co-express the chaperonin system GroEL and GroES from Oleispira antarctica. However, to achieve a large number of soluble target proteins, the expression conditions still need to be further optimized.

Optimization of culture conditions for the expression of three different insoluble proteins in Escherichia coli

Recombinant protein expression for structural and therapeutic applications requires the use of systems with high expression yields. Escherichia coli is considered the workhorse for this purpose, given its fast growth rate and feasible manipulation. However, bacterial inclusion body formation remains a challenge for further protein purification. We analyzed and optimized the expression conditions for three different proteins: an anti-MICA scFv, MICA, and p19 subunit of IL-23. We used a response surface methodology based on a three-level Box-Behnken design, which included three factors: post-induction temperature, post-induction time and IPTG concentration. Comparing this information with soluble protein data in a principal component analysis revealed that insoluble and soluble proteins have different optimal conditions for post-induction temperature, post-induction time, IPTG concentration and in amino acid sequence features. Finally, we optimized the refolding conditions of the least expressed protein, anti-MICA scFv, using a fast dilution protocol with different additives, obtaining soluble and active scFv for binding assays. These results allowed us to obtain higher yields of proteins expressed in inclusion bodies. Further studies using the system proposed in this study may lead to the identification of optimal environmental factors for a given protein sequence, favoring the acceleration of bioprocess development and structural studies.

Expression of DnaK and HtrA genes under high temperatures and their impact on thermotolerance of a Salmonella serotype isolated from tahini product

Background Salmonella is considered to be the second largest source of infection in food-borne diseases. It is also considered one of the most important dangers particularly in the meat and dairy industry. Therefore, the main objective of our study was to determine the relationship between thermotolerance of a Salmonella serotype and the expression of DnaK and HtrA genes. Results In this study, expression of the two genes DnaK and HtrA was compared under four different temperatures 37 °C, 42 °C, 50 °C, and 55 °C in two serotypes of Salmonella enterica subsp. enterica. One of them was isolated from tahini product and identified as Salmonella enterica subsp. enterica serovar choleraesuis. This identified serotype was found to be more thermotolerant than the second serotype (Salmonella enterica subsp. enterica serovar typhimurium (ATCC 13311)), which was used as reference. This conclusion was based on D and Z values, which were used to compare thermoresistance ability of the two serotypes under four different temperatures 60 °C, 65 °C, 70 °C, and 75 °C. In addition, the results of qRT-PCR showed that after 43 °C (induction temperature), the relative expression (fold change) of DnaK and HtrA genes increased up to 5 and 47, respectively, comparing to their expression at 37 °C. Conclusions Thermotolerance of the identified S. choleraesuis serotype showed significantly high expression levels of DnaK and HtrA genes.

Preparation of nano-SiO2 compound antioxidant and its antioxidant effect on polyphenylene sulfide

Polyphenylene sulfide (PPS) is easily oxidized at high temperature, which greatly limits its applications. In this study, a nano-SiO2 compound antioxidant (SiO2-g-AO) was prepared and incorporated into PPS by melt compounding to obtain PPS/SiO2-g-AO composites. SiO2-g-AO was prepared by reacting 3-(3,5-di-tert-butyl-4-hydroxyphenyl) (antioxidant AO) with an aminosilane coupling agent (KH792). Fourier transform infrared (FTIR) spectroscopy, energy dispersive spectroscopy (EDS) and thermogravimetric analysis (TGA) confirmed that the antioxidant AO was successfully immobilized on the surface of SiO2 and the thermal stability was improved. Scanning electron microscopy (SEM) images showed that SiO2-g-AO was uniformly dispersed in PPS. It has been found that the crystallinity and mechanical properties of PPS composites improved, and the dynamic oxidation induction temperature (OIT) increased in the range of 16.7°C–21.1°C. A synergistic anti-oxidation model of nanoparticles and antioxidants, namely a multi-stage antioxidant system, was established by comprehensive analysis of experimental results. The synergistic anti-oxidation model provides a new idea for the antioxidant modification of polymer composites.