scholarly journals Optimization of protein isolation by proteomic qualification from Cutaneotrichosporon oleaginosus

2019 ◽  
Vol 412 (2) ◽  
pp. 449-462 ◽  
Author(s):  
Dania Awad ◽  
Thomas Brueck
Keyword(s):  
Protein Extraction ◽  
Feedback Regulation ◽  
Genetic Optimization ◽  
Physiochemical Properties ◽  
High Lipid ◽  
Extraction Buffer ◽  
Optimized Protocol ◽  
Significant Step ◽  
One Step ◽  
Purification Methods

AbstractIn the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids.

scholarly journals Small Protein Enrichment Improves Proteomics Detection of sORF Encoded Polypeptides

2021 ◽  
Vol 12 ◽  
Author(s):  
Igor Fijalkowski ◽  
Marlies K. R. Peeters ◽  
Petra Van Damme
Keyword(s):  
Protein Extraction ◽  
Protein Solubility ◽  
Extraction Methods ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Physiochemical Properties ◽  
Small Proteins ◽  
Efficient Detection ◽  
Optimized Protocol ◽  
Small Open Reading Frames

With the rapid growth in the number of sequenced genomes, genome annotation efforts became almost exclusively reliant on automated pipelines. Despite their unquestionable utility, these methods have been shown to underestimate the true complexity of the studied genomes, with small open reading frames (sORFs; ORFs typically considered shorter than 300 nucleotides) and, in consequence, their protein products (sORF encoded polypeptides or SEPs) being the primary example of a poorly annotated and highly underexplored class of genomic elements. With the advent of advanced translatomics such as ribosome profiling, reannotation efforts have progressed a great deal in providing translation evidence for numerous, previously unannotated sORFs. However, proteomics validation of these riboproteogenomics discoveries remains challenging due to their short length and often highly variable physiochemical properties. In this work we evaluate and compare tailored, yet easily adaptable, protein extraction methodologies for their efficacy in the extraction and concomitantly proteomics detection of SEPs expressed in the prokaryotic model pathogen Salmonella typhimurium (S. typhimurium). Further, an optimized protocol for the enrichment and efficient detection of SEPs making use of the of amphipathic polymer amphipol A8-35 and relying on differential peptide vs. protein solubility was developed and compared with global extraction methods making use of chaotropic agents. Given the versatile biological functions SEPs have been shown to exert, this work provides an accessible protocol for proteomics exploration of this fascinating class of small proteins.

scholarly journals Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Shoji Suzuki ◽  
Norio Kurosawa
Keyword(s):  
Gene Knockout ◽  
Arginine Decarboxylase ◽  
Sulfolobus Acidocaldarius ◽  
Dna Photolyase ◽  
Genetic Studies ◽  
Multiple Gene ◽  
Plasmid Construction ◽  
Pcr Product ◽  
Optimized Protocol ◽  
One Step

Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA) by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER). In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr-) and arginine decarboxylase- (argD-) deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

scholarly journals Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis

2011 ◽  
Vol 5 ◽  
pp. BCBCR.S6263 ◽  
Author(s):  
Olena Zakharchenko ◽  
Christina Greenwood ◽  
Louise Alldridge ◽  
Serhiy Souchelnytskyi
Keyword(s):  
Gel Electrophoresis ◽  
Breast Tissue ◽  
Protein Extraction ◽  
Small Sample Size ◽  
Small Sample ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Main Challenge ◽  
Optimized Protocol ◽  
Separation Of Proteins

Proteomics is a highly informative approach to analyze cancer-associated transformation in tissues. The main challenge to use a tissue for proteomics studies is the small sample size and difficulties to extract and preserve proteins. The choice of a buffer compatible with proteomics applications is also a challenge. Here we describe a protocol optimized for the most efficient extraction of proteins from the human breast tissue in a buffer compatible with two-dimensional gel electrophoresis (2D-GE). This protocol is based on mechanically assisted disintegration of tissues directly in the 2D-GE buffer. Our method is simple, robust and easy to apply in clinical practice. We demonstrate high quality of separation of proteins prepared according to the reported here protocol.

scholarly journals An Experimental Investigation of Karanja Biodiesel Production in Sarawak, Malaysia

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Dewi Harreh ◽  
A. A. Saleh ◽  
A. N. R. Reddy ◽  
S. Hamdan
Keyword(s):  
Experimental Investigation ◽  
Clean Energy ◽  
Biodiesel Production ◽  
Brake Specific Fuel Consumption ◽  
Physiochemical Properties ◽  
Brake Thermal Efficiency ◽  
The Past ◽  
Brake Power ◽  
One Step ◽  
Karanja Oil

The application of nonedible feedstock for the production of biodiesel has become an area of research interest among clean energy experts in the past few years. This research is aimed at the utilization of Pongamia pinnata (karanja), a nonedible feedstock from the state of Sarawak, Malaysia, to produce biodiesel to be known as crude karanja oil (CKO). A one-step transesterification process utilizing 7 : 1–10 : 1 wt% methanol (CH3OH) and 0.5–1.2 wt% sodium hydroxide (NaOH) at 65°C for 1.5 hrs has been used for the biodiesel production yielding 84% conversion. The physiochemical properties of the CKO produced revealed that it conforms with EN14214 standards for brake power (BP), brake specific fuel consumption (BSFC), and brake thermal efficiency (BTE) as they are all noted be optimal at B40.

scholarly journals Dramatic Improvement of Proteomic Analysis of Zebrafish Liver Tumor by Effective Protein Extraction with Sodium Deoxycholate and Heat Denaturation

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Jigang Wang ◽  
Yew Mun Lee ◽  
Caixia Li ◽  
Ping Li ◽  
Zhen Li ◽  
...  
Keyword(s):  
Liver Tumor ◽  
Proteomic Analysis ◽  
Protein Extraction ◽  
Sodium Deoxycholate ◽  
Heat Denaturation ◽  
Dramatic Improvement ◽  
Tissue Samples ◽  
Highly Efficient ◽  
Extraction Buffer ◽  
Zebrafish Liver

Majority of the proteomic studies on tissue samples involve the use of gel-based approach for profiling and digestion. The laborious gel-based approach is slowly being replaced by the advancing in-solution digestion approach. However, there are still several difficulties such as difficult-to-solubilize proteins, poor proteomic analysis in complex tissue samples, and the presence of sample impurities. Henceforth, there is a great demand to formulate a highly efficient protein extraction buffer with high protein extraction efficiency from tissue samples, high compatibility with in-solution digestion, reduced number of sample handling steps to reduce sample loss, low time consumption, low cost, and ease of usage. Here, we evaluated various existing protein extraction buffers with zebrafish liver tumor samples and found that sodium deoxycholate- (DOC-) based extraction buffer with heat denaturation was the most effective approach for highly efficient extraction of proteins from complex tissues such as the zebrafish liver tumor. A total of 4,790 proteins have been identified using shotgun proteomics approach with 2D LC, which to our knowledge is the most comprehensive study for zebrafish liver tumor proteome.

scholarly journals Mesoporous Silica with Covalently Immobilized Anthracene as Adsorbent for SPE Recovery of PAHs Pollutants from Highly Lipidic Solutions

2020 ◽  
Author(s):  
Albina Mikhraliieva ◽  
Rodrigo Araújo Gonçalves ◽  
Volodymyr Zaitsev
Keyword(s):  
Mesoporous Silica ◽  
Vegetable Oil ◽  
Myristic Acid ◽  
Polar Solvent ◽  
Dynamic Adsorption ◽  
Functionalized Mesoporous Silica ◽  
High Lipid ◽  
Two Samples ◽  
One Step ◽  
Π Stacking Interaction

Two samples of functionalized mesoporous silica containing anchored anthrylmethylamine groups (SiO<sub>2</sub>-Ant) have been prepared by surface assembling (1) and one step silane immobilization (2). Both adsorbents can be attributed to bimodal balanced hydrophobic-hydrophilic adsorbents with loading of anthracene groups about 15-33%. The adsorbents have been used for SPE of anthracene from organic solvents (acetonitrile, acetone and heptane) and model solutions of lipids (myristic acid and vegetable oil). The obtained results were compared with commercial C18 SPE cartridge. While C18 cartridge recovers anthracene from water-containing media (acetonitrile/water, 1/1), SiO<sub>2</sub>-Ant cartridges much more efficient in extraction of anthracene from non-polar solvent (heptane). Lipids macrocomponents such as myristic acid and vegetable oil do not decrease the dynamic adsorption capacity and recovery of the model PAH on SiO<sub>2</sub>-Ant. It was demonstrated that π-π stacking interaction with the analyte determine the selectivity of SiO<sub>2</sub>-Ant towards of anthracene. This makes SiO<sub>2</sub>-Ant attractive for selective pre-concentration of PAHs from high lipid content objects, such as vegetable oils.

scholarly journals Mesoporous Silica with Covalently Immobilized Anthracene as Adsorbent for SPE Recovery of PAHs Pollutants from Highly Lipidic Solutions

2020 ◽  
Author(s):  
Albina Mikhraliieva ◽  
Rodrigo Araújo Gonçalves ◽  
Volodymyr Zaitsev
Keyword(s):  
Mesoporous Silica ◽  
Vegetable Oil ◽  
Myristic Acid ◽  
Polar Solvent ◽  
Dynamic Adsorption ◽  
Functionalized Mesoporous Silica ◽  
High Lipid ◽  
Two Samples ◽  
One Step ◽  
Π Stacking Interaction

Two samples of functionalized mesoporous silica containing anchored anthrylmethylamine groups (SiO<sub>2</sub>-Ant) have been prepared by surface assembling (1) and one step silane immobilization (2). Both adsorbents can be attributed to bimodal balanced hydrophobic-hydrophilic adsorbents with loading of anthracene groups about 15-33%. The adsorbents have been used for SPE of anthracene from organic solvents (acetonitrile, acetone and heptane) and model solutions of lipids (myristic acid and vegetable oil). The obtained results were compared with commercial C18 SPE cartridge. While C18 cartridge recovers anthracene from water-containing media (acetonitrile/water, 1/1), SiO<sub>2</sub>-Ant cartridges much more efficient in extraction of anthracene from non-polar solvent (heptane). Lipids macrocomponents such as myristic acid and vegetable oil do not decrease the dynamic adsorption capacity and recovery of the model PAH on SiO<sub>2</sub>-Ant. It was demonstrated that π-π stacking interaction with the analyte determine the selectivity of SiO<sub>2</sub>-Ant towards of anthracene. This makes SiO<sub>2</sub>-Ant attractive for selective pre-concentration of PAHs from high lipid content objects, such as vegetable oils.

Optimized expression of recombinant human NIMA-related kinase 7 (NEK7) with a higher purity in Escherichia coli

2021 ◽  
Vol 28 ◽  
Author(s):  
Xing-Jie Zhang ◽  
Ting-Ting Wang ◽  
Yu-Kun Pu ◽  
Lin Zeng ◽  
Rui-Han Zhang ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Inflammatory Diseases ◽  
Gel Filtration ◽  
Serine Threonine Kinase ◽  
E Coli ◽  
Threonine Kinase ◽  
His Tag ◽  
Optimized Protocol ◽  
One Step ◽  
Affinity Resin

Background: NIMA (never in mitosis, gene A) serine/threonine kinase 7 (NEK7) is a regulator of mitosis spindle in mammals and is considered as a drug target of inflammasome related inflammatory diseases. However, most commercially available or reported recombinant NEK7 proteins are either inactive or have low purity. These shortcomings limit the pharmacological studies and development of NEK7 inhibitors. Objective: To elucidate what causes the NEK7 low purity in E. coli, and optimize a protocol to improve the protein purity. Methods: A comparative study of expression full length NEK7 with an N-terminal His-tag or a Cterminal His-tag was performed. His-affinity resin, ion exchange and gel filtration chromatography were used to purify NEK7. The protein was identified by mass spectrometry. The activity and folding of NEK7 were evaluated by chemiluminescent assay and thermal shift assay. Results: Our results demonstrated that N-terminal tagged protein was toxic to E. coli, resulting in incomplete translated products. The C-terminal tagged NEK7-His6 had a much higher purity than that of an N-terminal tag. The Ni2+ resin one-step purification led to a purity of 91.7%, meeting the criteria of most kinase assays. With two-step and three-step procedures, the protein purities were 94.7% and ~100%, respectively. The NEK7 purified in this work maintained its kinase activity and correct conformation, and the compound-protein interaction ability. Conclusion: Our optimized protocol could produce good purity of His tagged NEK7 in E. coli, and the kinase activity and biophysical characteristics of which are preserved.

Microwave-assisted synthesis of the anticancer drug cisplatin, cis-[Pt(NH3)2Cl2]

2015 ◽  
Vol 44 (7) ◽  
pp. 3384-3392 ◽  
Author(s):  
Emanuele Petruzzella ◽  
Cristian V. Chirosca ◽  
Cameron S. Heidenga ◽  
James D. Hoeschele
Keyword(s):  
Anticancer Drug ◽  
Microwave Assisted Synthesis ◽  
Microwave Assisted ◽  
One Step ◽  
Purification Methods

A rapid one-step microwave-assisted synthesis of cisplatin and two purification methods were developed. The process requires about 80 min and could be used to incorporate short-lived Pt radionuclides into cisplatin.

Sign in / Sign up

Export Citation Format

Share Document