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https://www.readbyqxmd.com/read/29785668/hac1p-homologues-from-higher-eukaryotes-can-improve-the-secretion-of-heterologous-proteins-in-the-yeast-pichia-pastoris
#1
Olufemi Emmanuel Bankefa, Meiyu Wang, Taicheng Zhu, Yin Li
OBJECTIVES: To systematically explore the effects of overexpressing Hac1p homologues from different sources on protein secretion in Pichia pastoris system. RESULTS: Effects of Hac1p homologues encompassing P. pastoris (PpHac1p), S. cerevisiae (ScHac1p), Trichoderma reesei (TrHac1p) and Homo sapiens (HsXbp1), on secretion of three reporter proteins-β-galactosidase, β-mannanase and glucose oxidase were investigated. No individual Hac1p was optimal for all the enzymes...
May 21, 2018: Biotechnology Letters
https://www.readbyqxmd.com/read/29779067/cost-effective-large-scale-expression-of-proteins-for-nmr-studies
#2
Julia Klopp, Aurélie Winterhalter, Rémy Gébleux, Daniela Scherer-Becker, Christian Ostermeier, Alvar D Gossert
We present protocols for high-level expression of isotope-labelled proteins in E. coli in cost-effective ways. This includes production of large amounts of unlabeled proteins and 13 C-methyl methionine labeling in rich media, where yields of up to a gram of soluble protein per liter of culture are reached. Procedures for uniform isotope labeling of 2 H, 13 C and 15 N using auto-induction or isopropyl-β-D-1-thiogalactopyranoside-induction are described, with primary focus on minimal isotope consumption and high reproducibility of protein expression...
May 19, 2018: Journal of Biomolecular NMR
https://www.readbyqxmd.com/read/29765812/enhanced-production-and-purification-of-recombinant-surface-array-protein-sap-for-use-in-detection-of-bacillus-anthracis
#3
Nidhi Puranik, N K Tripathi, V Pal, Ajay Kumar Goel
Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively...
May 2018: 3 Biotech
https://www.readbyqxmd.com/read/29753122/high-level-expression-and-glycosylation-of-recombinant-mycobacterium-tuberculosis-ala-pro-rich-antigen-in-pichia-pastoris
#4
Shengjun Wang, Yaoguang Wang, Peng George Wang, Min Chen, Yun Kong
The Ala-Pro-rich Antigen (Apa) from Mycobacterium tuberculosis is a mannosylated protein with immunogenic and antigenic properties. The O-mannosylation is essential for its biological function in the process of colonization and invasion of host cells by M. tuberculosis. In this work, the gene encoding Apa was cloned from M. tuberculosis and expressed in Pichia pastoris GS115. In shake-flasks, the recombinant Apa was secreted into the culture media and purified with a yield of 0.6 g/L. Both N- and O-glycans were found in recombinant Apa...
May 9, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29750118/improved-microscale-cultivation-of-pichia-pastoris-for-clonal-screening
#5
Alexander Eck, Matthias Schmidt, Stefanie Hamer, Anna Joelle Ruff, Jan Förster, Ulrich Schwaneberg, Lars M Blank, Wolfgang Wiechert, Marco Oldiges
Background: Expanding the application of technical enzymes, e.g., in industry and agriculture, commands the acceleration and cost-reduction of bioprocess development. Microplates and shake flasks are massively employed during screenings and early phases of bioprocess development, although major drawbacks such as low oxygen transfer rates are well documented. In recent years, miniaturization and parallelization of stirred and shaken bioreactor concepts have led to the development of novel microbioreactor concepts...
2018: Fungal Biology and Biotechnology
https://www.readbyqxmd.com/read/29744279/pathway-engineering-in-corynebacterium-glutamicum-s9114-for-5-aminolevulinic-acid-production
#6
Bin Zhang, Bang-Ce Ye
5-Aminolevulinic acid (ALA) is a non-protein amino acid with a significant potential for cancer treatment and plant stress resistance. Microbial fermentation has gradually replaced the traditional chemical-based method for ALA production, thus increasing the need for high-ALA-producing strains. In this study, we engineered the glutamate producing strain, Corynebacterium glutamicum S9114, for ALA production. To efficiently convert l-glutamate to ALA, hemA and hemL from Salmonella typhimurium and Escherichia coli were tandemly overexpressed...
May 2018: 3 Biotech
https://www.readbyqxmd.com/read/29723667/the-missing-piece-in-the-puzzle-prediction-of-aggregation-via-the-protein-protein-interaction-parameter-a-%C3%A2-2
#7
Ellen Koepf, Rudolf Schroeder, Gerald Brezesinski, Wolfgang Friess
The tendency of protein pharmaceuticals to form aggregates is a major challenge during formulation development, as aggregation affects quality and safety of the product. In particular, the formation of large native-like particles in the context of liquid-air interfacial stress is a well-known but not fully understood problem. Focusing on the two most fundamental criteria of protein formulation affecting protein-protein interaction, the impact of pH and ionic strength on the interaction parameter A∗ 2 and its link to aggregation upon mechanical stress was investigated...
April 30, 2018: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/29696339/yarrowia-lipolytica-morphological-mutant-enables-lasting-in-situ-immobilization-in-bioreactor
#8
Marie Vandermies, Tambi Kar, Frédéric Carly, Jean-Marc Nicaud, Frank Delvigne, Patrick Fickers
In the present study, we have isolated and characterized a Yarrowia lipolytica morphological mutant growing exclusively in the pseudohyphal morphology. The gene responsible for this phenotype, YALI0E06519g, was identified as homologous to the mitosis regulation gene HSL1 from Saccharomyces cerevisiae. Taking advantage of its morphology, we achieved the immobilization of the Δhsl1 mutant on the metallic structured packing of immobilized-cell bioreactors. We obtained significant cell retention and growth on the support during shake flask and bioreactor experiments without an attachment step prior to the culture...
April 26, 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29679607/process-intensification-of-core-streptavidin-production-through-high-cell-density-cultivation-of-recombinant-e-coli-and-a-temperature-based-refolding-method
#9
Leong Huat Chua, Soo Choon Tan, Mervyn W O Liew
An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1 ) was obtained from induction at OD 100 without significant reductions in specific yield...
April 18, 2018: Journal of Biotechnology
https://www.readbyqxmd.com/read/29675805/enhanced-extracellular-pullulanase-production-in-bacillus-subtilis-using-protease-deficient-strains-and-optimal-feeding
#10
Kang Zhang, Lingqia Su, Jing Wu
To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6-WS11, respectively. The strains WS3 (none) and WS4 (ΔnprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3-WS11, the pullulanase production levels of the resulting strains (WS3PUL-WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148...
April 20, 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29670331/cell-penetrating-peptide-conjugates-of-gambogic-acid-enhance-the-antitumor-effect-on-human-bladder-cancer-ej-cells-through-ros-mediated-apoptosis
#11
Lei Lyu, Lu-Qi Huang, Tao Huang, Wei Xiang, Jing-Dong Yuan, Chuan-Hua Zhang
Background: Gambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs. Purpose: The object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells...
2018: Drug Design, Development and Therapy
https://www.readbyqxmd.com/read/29660471/efficient-production-of-human-goose-type-lysozyme-2-in-the-methylotrophic-yeast-pichia-pastoris
#12
Peng Huang, Qingwen Sun, Weijun Shi, Wangchun Du, Xue Li, Ning Zhang
Infectious diseases caused by antibiotic multidrug-resistant microorganisms are major causes of morbidity and mortality in humans. Hence, there is an urgent need to search for new antimicrobial agents. Initially known as a defensive effector in the innate immunity of certain organs of the human body, human goose-type lysozyme 2 (hLysG2) has been shown to possess therapeutically useful potential against multidrug-resistant microorganisms. Developing a novel strategy for large-scale production that provides high yields of this protein with high purity, quality, and potency is critical for pharmaceutical applications...
April 13, 2018: Journal of Biotechnology
https://www.readbyqxmd.com/read/29622042/engineering-crispr-interference-system-in-klebsiella-pneumoniae-for-attenuating-lactic-acid-synthesis
#13
Jingxuan Wang, Peng Zhao, Ying Li, Lida Xu, Pingfang Tian
BACKGROUND: Klebsiella pneumoniae is a promising industrial species for bioproduction of bulk chemicals such as 1,3-propanediol, 2,3-butanediol and 3-hydroxypropionic acid (3-HP). However, lactic acid is a troublesome by-product when optimizing for 3-HP production. Therefore, it is highly desirable to minimize lactic acid. RESULTS: Here, we show that lactic acid synthesis can be largely blocked by an engineered CRISPR interference (CRISPRi) system in K. pneumoniae...
April 5, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29619083/engineering-of-e-coli-inherent-fatty-acid-biosynthesis-capacity-to-increase-octanoic-acid-production
#14
Zaigao Tan, Jong Moon Yoon, Anupam Chowdhury, Kaitlin Burdick, Laura R Jarboe, Costas D Maranas, Jacqueline V Shanks
Background: As a versatile platform chemical, construction of microbial catalysts for free octanoic acid production from biorenewable feedstocks is a promising alternative to existing petroleum-based methods. However, the bio-production strategy has been restricted by the low capacity of E. coli inherent fatty acid biosynthesis. In this study, a combination of integrated computational and experimental approach was performed to manipulate the E. coli existing metabolic network, with the objective of improving bio-octanoic acid production...
2018: Biotechnology for Biofuels
https://www.readbyqxmd.com/read/29614035/investigating-the-nutritional-and-recovery-habits-of-tennis-players
#15
James A Fleming, Robert J Naughton, Liam D Harper
In this study, the nutritional and recovery habits of tennis players pre-, during, and post-match-play were investigated. Seventy tennis players completed a bespoke nutrition and recovery habits questionnaire, with questions related to the following areas: match preparation, intra-match nutritional habits, situation dependent variables, and post-match nutrition and recovery. On match day-1, the consumption of balanced meals consisting of carbohydrate (CHO), fat and protein, with some micronutrient considerations were reported by 51% of players...
April 3, 2018: Nutrients
https://www.readbyqxmd.com/read/29601964/a-systematic-analysis-of-vrc01-antibody-expression-in-pichia-pastoris-through-signal-peptide-optimization
#16
Rochelle Aw, Paul F McKay, Robin J Shattock, Karen M Polizzi
Pichia (syn. Komagataella) pastoris has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6...
March 27, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29596634/expression-and-purification-of-biologically-active-recombinant-rabbit-monocyte-chemoattractant-protein1-in-escherichia-coli
#17
Maryam Boshtam, Hossein Khanahmad Shahreza, Sadegh Feizollahzadeh, Ilnaz Rahimmanesh, Seddigheh Asgary
Monocyte chemoattractant protein 1 (MCP1) with recruiting monocytes is an important factor at the beginning of inflammatory disorders such as atherosclerosis which seems its blocking preclude this process and help improvement of related diseases. To perform clinical research in this field, MCP1 protein is required but firstly, animal studies should be done. As the rabbit is a suitable model for many inflammatory disorders, and Escherichia coli BL21(DE3) (BL21) cell is a high-efficiency host for protein expression, we decided to produce recombinant rabbit MCP1 (rRMCP1) in BL21/pET28a system...
March 27, 2018: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/29555311/enhanced-production-of-soluble-tumor-necrosis-factor-related-apoptosis-inducing-ligand-in-escherichia-coli-using-a-novel-self-cleavable-tag-system-fh8-%C3%AE-i-cm
#18
Min Zhang, Zhanqing Wang, Lili Chi, Jing Sun, Yaling Shen
Escherichia coli is an essential host for large-scale expression of heterologous polypeptides. However, further applications are limited by the formation of potential protein aggregates. In this work, we developed a novel on-column tag removal and purification system based on Fh8 hydrophobic interaction chromatography purification and ΔI-CM self-cleavage to obtain soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We evaluated several methods to improve TRAIL solubility and finally demonstrated that the Fh8 tag was a powerful solubility enhancer...
March 16, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29519516/characterization-and-functional-analysis-of-pnuc-that-is-involved-in-the-oxidative-stress-tolerance-and-virulence-of-streptococcus-suis-serotype-2
#19
Quan Li, Yuhang Zhang, Du Dechao, Yu Yanfei, Wei Zhang
Streptococcus suis, an important swine pathogen and a major zoonotic agent, is responsible for severe financial losses in the global swine industry. Although a multitude of virulence factors have been reported, the pathogenesis of S. suis infections remains poorly understood. In our previous work, we identified a potential virulence-associated protein, named PnuC, unique to virulent strains of S. suis serotype 2 (S. suis 2). To investigate the functions of PnuC, the pnuC gene deletion mutant (ΔpnuC) was constructed in S...
March 2018: Veterinary Microbiology
https://www.readbyqxmd.com/read/29518537/high-level-expression-and-purification-of-recombinant-human-serum-albumin-in-pichia-pastoris
#20
Wen Zhu, Guihua Gong, Jie Pan, Shu Han, Wei Zhang, Youjia Hu, Liping Xie
Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418...
July 2018: Protein Expression and Purification
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