scholarly journals Secretory Expression Of The Glucan Endo-1,3-Beta-D-Glucosidase Gene Of Secale Cereale In Yeast Pichia Pastoris

2021 ◽  
Author(s):  
M. Saginova ◽  
Zh. Akishev ◽  
A. Sarsen ◽  
A. Kiribayeva ◽  
B. Khassenov
Keyword(s):  
Pichia Pastoris ◽  
Secale Cereale ◽  
Culture Medium ◽  
Freezing Point ◽  
Shuttle Vector ◽  
Antifreeze Proteins ◽  
Yeast Pichia Pastoris ◽  
Adaptive Mechanisms ◽  
Living Organisms ◽  
Almost All

For survival in cold conditions, many organisms have developed unique adaptive mechanisms based on the synthesis of antifreeze proteins, peptides and glycoproteins that prevent ice formation at negative temperatures. These molecules tend to bind ice crystals and lower the freezing point of the solution without the formation of large crystals. Antifreeze proteins (AFP) were found in almost all types of living organisms, including insects, fungus, yeasts, bacteria and plants. The gene of antifreeze protein - glucan endo-1,3-beta-D-glucosidase (ScGlu-3) from Secale cereale was cloned into shuttle vector pPICZαA. The competent cells of yeast Pichia pastoris GS115 were transformed and the producer strain was obtained, which secreted of ScGlu-3 into the culture medium using 3% methanol as the only carbon source. It was found by western blotting that the maximum accumulation of ScGlu-3 in the culture occurs after 48 hours of fermentation on a medium with methanol. Established that rScGlu-3 precipitates at 50-65% of ammonium sulfate.

HVEM Cryomicroscopy of Frozen Hydrated PTK1 Cells

1990 ◽  
Vol 48 (1) ◽  
pp. 500-501
Author(s):  
E.T. O’Toole ◽  
G.P. Wray ◽  
J.R. Kremer ◽  
J.R. Mcintosh
Keyword(s):  
Liquid Nitrogen ◽  
Phase Contrast ◽  
Culture Medium ◽  
Low Dose ◽  
Ammonium Acetate ◽  
Freezing Point ◽  
Voltage Electron ◽  
Cold Stage ◽  
Humidity Chamber ◽  
Coated Carbon

Ultrarapid freezing and cryomicroscopy of frozen hydrated material makes it possible to visualize samples that have never been exposed to chemical fixatives, dehydration, or stains. In principle, freezing and cryoimaging methods avoid artifacts associated with chemical fixation and processing and allow one to visualize the specimen in a condition that is close to its native state. Here we describe a way to use a high voltage electron microscope (HVEM) for the cryoimaging of frozen hydrated PTK1 cells.PTK1 cells were cultured on formvar-coated, carbon stabilized gold grids. After three days in culture, the grids were removed from the culture medium and blotted in a humidity chamber at 35° C. In some instances, the grids were rinsed briefly in 0.16 M ammonium acetate buffer (pH 7.2) prior to blotting. After blotting, the grids were transferred to a plunging apparatus and plunged into liquid ethane held directly above its freezing point. The plunging apparatus consists of a vertical slide rail that guides the fall of a mounted pair of forceps that clamp the specimen. The forceps are surrounded by a plexiglass humidity chamber mounted over a dewar of liquid nitrogen containing an ethane chamber. After freezing, the samples were transferred to liquid nitrogen and viewed in a JEOL JEM 1000 equipped with a top entry cold stage designed and built by Mr. George Wray (Univ. Colorado). The samples were routinely exposed to electron doses of 1 e/Å2/sec, and viewed at a temperature of −150° C. A GATAN video system was used to enhance contrast and to estimate the correct amount of underfocus needed to obtain phase contrast at various magnifications. Low dose micrographs were taken using two second exposures of Kodak 4463 film. The state of the solid water in the specimen was determined by diffraction using a 30/μm field limiting aperture and a camera length of 1 meter.

iAFP-gap-SMOTE: An Efficient Feature Extraction Scheme Gapped Dipeptide Composition is Coupled with an Oversampling Technique for Identification of Antifreeze Proteins

2019 ◽  
Vol 16 (4) ◽  
pp. 294-302 ◽  
Author(s):  
Shahid Akbar ◽  
Maqsood Hayat ◽  
Muhammad Kabir ◽  
Muhammad Iqbal
Keyword(s):  
Feature Extraction ◽  
Amino Acid ◽  
Antifreeze Proteins ◽  
Protein Sequences ◽  
Sampling Technique ◽  
Lower Class ◽  
Success Rates ◽  
Throughput Model ◽  
Extraction Scheme ◽  
Living Organisms

Antifreeze proteins (AFPs) perform distinguishable roles in maintaining homeostatic conditions of living organisms and protect their cell and body from freezing in extremely cold conditions. Owing to high diversity in protein sequences and structures, the discrimination of AFPs from non- AFPs through experimental approaches is expensive and lengthy. It is, therefore, vastly desirable to propose a computational intelligent and high throughput model that truly reflects AFPs quickly and accurately. In a sequel, a new predictor called “iAFP-gap-SMOTE” is proposed for the identification of AFPs. Protein sequences are expressed by adopting three numerical feature extraction schemes namely; Split Amino Acid Composition, G-gap di-peptide Composition and Reduce Amino Acid alphabet composition. Usually, classification hypothesis biased towards majority class in case of the imbalanced dataset. Oversampling technique Synthetic Minority Over-sampling Technique is employed in order to increase the instances of the lower class and control the biasness. 10-fold cross-validation test is applied to appraise the success rates of “iAFP-gap-SMOTE” model. After the empirical investigation, “iAFP-gap-SMOTE” model obtained 95.02% accuracy. The comparison suggested that the accuracy of” iAFP-gap-SMOTE” model is higher than that of the present techniques in the literature so far. It is greatly recommended that our proposed model “iAFP-gap-SMOTE” might be helpful for the research community and academia.

scholarly journals Adsorption process and mechanism of heavy metal ions by different components of cells, using yeast (Pichia pastoris) and Cu2+ as biosorption models

RSC Advances ◽  
2021 ◽  
Vol 11 (28) ◽  
pp. 17080-17091
Author(s):  
Xinggang Chen ◽  
Zhuang Tian ◽  
Haina Cheng ◽  
Gang Xu ◽  
Hongbo Zhou
Keyword(s):  
Heavy Metal ◽  
Pichia Pastoris ◽  
Metal Ions ◽  
Cell Walls ◽  
Heavy Metal Ions ◽  
Adsorption Process ◽  
Yeast Pichia Pastoris ◽  
Adsorption Sites

The Cu2+ first bound to the outer mannan and finally entered the cytoplasm. During the whole adsorption process, the number of adsorption sites in the outer and middle cell walls was the largest, and then gradually decreased.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

scholarly journals The Diverse Calpain Family in Trypanosomatidae: Functional Proteins Devoid of Proteolytic Activity?

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 299
Author(s):  
Vítor Ennes-Vidal ◽  
Marta Helena Branquinha ◽  
André Luis Souza dos Santos ◽  
Claudia Masini d’Avila-Levy
Keyword(s):  
Proteolytic Activity ◽  
Current Knowledge ◽  
Current Therapy ◽  
Calcium Dependent ◽  
Administration Routes ◽  
Life Threatening ◽  
Living Organisms ◽  
Almost All ◽  
Open Question ◽  
Calpain Inhibitors

Calpains are calcium-dependent cysteine peptidases that were originally described in mammals and, thereafter, their homologues were identified in almost all known living organisms. The deregulated activity of these peptidases is associated with several pathologies and, consequently, huge efforts have been made to identify selective inhibitors. Trypanosomatids, responsible for life-threatening human diseases, possess a large and diverse family of calpain sequences in their genomes. Considering that the current therapy to treat trypanosomatid diseases is limited to a handful of drugs that suffer from unacceptable toxicity, tough administration routes, like parenteral, and increasing treatment failures, a repurposed approach with calpain inhibitors could be a shortcut to successful chemotherapy. However, there is a general lack of knowledge about calpain functions in these parasites and, currently, the proteolytic activity of these proteins is still an open question. Here, we highlight the current research and perspectives on trypanosomatid calpains, overview calpain description in these organisms, and explore the potential of targeting the calpain system as a therapeutic strategy. This review gathers the current knowledge about this fascinating family of peptidases as well as insights into the puzzle: are we unable to measure calpain activity in trypanosomatids, or are the functions of these proteins devoid of proteolytic activity in these parasites?

scholarly journals Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1379-1391
Author(s):  
Monique A Johnson ◽  
Hans R Waterham ◽  
Galyna P Ksheminska ◽  
Liubov R Fayura ◽  
Joan Lin Cereghino ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Allyl Alcohol ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Direct Selection ◽  
Toxic Exposure ◽  
Peroxisome Biogenesis ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris ◽  
Selection Of

Abstract We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.

scholarly journals Comparison on the Nutrient Plunder Capacity of Orychophragmus violaceus and Brassica napus L. Based on Electrophysiological Information

Horticulturae ◽  
2021 ◽  
Vol 7 (8) ◽  
pp. 206
Author(s):  
Cheng Zhang ◽  
Yue Su ◽  
Yanyou Wu ◽  
Haitao Li ◽  
Ying Zhou ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Orychophragmus Violaceus ◽  
Nutrient Metabolism ◽  
Brassica Napus L ◽  
Adaptive Mechanisms ◽  
Capacitive Reactance ◽  
Time Determination ◽  
Almost All ◽  
First Time

The nutrient metabolism, growth and development of plants are strongly affected by its nutrient plunder, and plants have different adaptive mechanisms to low-nutrient environments. The electrophysiological activities involve almost all life processes of plants. In this study, the active transport flow of nutrient (NAF) and nutrient plunder capacity (NPC) of plants were defined based on leaf intrinsic impedance (IZ), capacitive reactance (IXc), inductive reactance (IXL) and capacitance (IC) to evaluate the nutrient plunder capacity of plants for the first time. The results indicate that Orychophragmus violaceus had higher (p < 0.01) NPC and IC and lower (p < 0.01) IR, IXc, IXL and IZ as compared to Brassica napus L., which supports a superior ion affinity and that it could be better adapted to low-nutrient environments. UAF and NPC of plants exhibited good correlations with crude protein, crude ash and water content, and precisely revealed the plunder capacity and adaptive strategies of plants to nutrients. The present work highlights that O. violaceus had superior NPC and ion affinity compared with B. napus, and provided a novel, rapid, reliable method based on the plant’s electrophysiological information for real-time determination of the nutrient plunder capacity of plants.

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