In vivo and in vitro protein solubility assays using split GFP

2006 ◽  
Vol 3 (10) ◽  
pp. 845-854 ◽  
Author(s):  
Stéphanie Cabantous ◽  
Geoffrey S Waldo
Keyword(s):  
Protein Solubility ◽  
Vitro Protein

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Effect of Processing on Fish Protein Antigenicity and Allergenicity

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 969
Author(s):  
Xingyi Jiang ◽  
Qinchun Rao
Keyword(s):  
Fish Species ◽  
Protein Denaturation ◽  
Ex Vivo ◽  
Protein Solubility ◽  
Future Research ◽  
Food Protein ◽  
In Vivo Evaluation ◽  
Fish Allergy

Fish allergy is a life-long food allergy whose prevalence is affected by many demographic factors. Currently, there is no cure for fish allergy, which can only be managed by strict avoidance of fish in the diet. According to the WHO/IUIS Allergen Nomenclature Sub-Committee, 12 fish proteins are recognized as allergens. Different processing (thermal and non-thermal) techniques are applied to fish and fishery products to reduce microorganisms, extend shelf life, and alter organoleptic/nutritional properties. In this concise review, the development of a consistent terminology for studying food protein immunogenicity, antigenicity, and allergenicity is proposed. It also summarizes that food processing may lead to a decrease, no change, or even increase in fish antigenicity and allergenicity due to the change of protein solubility, protein denaturation, and the modification of linear or conformational epitopes. Recent studies investigated the effect of processing on fish antigenicity/allergenicity and were mainly conducted on commonly consumed fish species and major fish allergens using in vitro methods. Future research areas such as novel fish species/allergens and ex vivo/in vivo evaluation methods would convey a comprehensive view of the relationship between processing and fish allergy.

scholarly journals In Vivo and In Vitro Protein Ligation by Naturally Occurring and Engineered Split DnaE Inteins

PLoS ONE ◽  
2009 ◽  
Vol 4 (4) ◽  
pp. e5185 ◽  
Author(s):  
A. Sesilja Aranko ◽  
Sara Züger ◽  
Edith Buchinger ◽  
Hideo Iwaï
Keyword(s):  
Protein Ligation ◽  
Naturally Occurring ◽  
Vitro Protein

Use of an in Vitro Protein Synthesizing System to Test the Mode of Action of Chloracetamides

Weed Science ◽  
1980 ◽  
Vol 28 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Luanne M. Deal ◽  
J. T. Reeves ◽  
B. A. Larkins ◽  
F. D. Hess
Keyword(s):  
Protein Synthesis ◽  
Sand Culture ◽  
Leucine Incorporation ◽  
Insoluble Protein ◽  
In Vitro System ◽  
A Cell ◽  
Protein Incorporation ◽  
Vitro Protein

The effects of chloracetamides on protein synthesis were studied both in vivo and in vitro. Four chloracetamide herbicides, alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide], metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl)acetamide], CDAA (N–N-diallyl-2-chloroacetamide), and propachlor (2-chloro-N-isopropylacetanilide) were tested for inhibition of [3H]-leucine incorporation into protein. Incorporation of3H-leucine into trichloroacetic acid (TCA)-insoluble protein was inhibited in oat (Avena sativaL. ‘Victory’) seedlings grown in sand culture and treated 12 h at 1 × 10−4M with these chloracetamides. The herbicides were also tested in a cell-free protein synthesizing system containing polyribosomes purified from oat root cytoplasm. These herbicides had no effect on the rates of polypeptide elongation nor on the synthesis of specific polypeptides when herbicides (1 × 10−4M) were added directly to the system. Polypeptide formation was inhibited 89% when 1 × 10−4M cycloheximide was added during translation. Cytoplasmic polyribosomes were isolated from oat roots treated 12 h with 1 × 10−4M herbicide. Translation rates and products were not altered when these polyribosomes were added to the in vitro system. Protein synthesis is inhibited when tested in an in vivo system; however, the inhibition does not occur during the translation of mRNA into protein.

scholarly journals Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis

2016 ◽  
Vol 206 (1) ◽  
pp. 11-22 ◽  
Author(s):  
P. Martijn den Reijer ◽  
Marjan Sandker ◽  
Susan V. Snijders ◽  
Mehri Tavakol ◽  
Antoni P. A. Hendrickx ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Detection ◽  
Antibody Detection ◽  
Potential Vaccine ◽  
Vitro Protein

Effects of Fermentation on Nutritional and Functional Properties of Soybean, Maize, and Germinated Sorghum Composite Flour

2012 ◽  
Vol 8 (1) ◽  
Author(s):  
Nicole Murekatete ◽  
Yufei Hua ◽  
Xiangzhen Kong ◽  
Caimeng Zhang
Keyword(s):  
Protein Solubility ◽  
Titratable Acidity ◽  
Protein Digestibility ◽  
Composite Flour ◽  
In Vitro Protein Digestibility ◽  
Food Ingredient ◽  
Fermentation Time ◽  
Nutrient Density ◽  
Vitro Protein

Sorghum germination resulted in a substantial tannin loss (95.7 %). Proximate composition, titratable acidity, pasting properties, in vitro protein digestibility, and protein solubility were studied post fermentation (Saccharomyces Cerevisiae) of the blended soybean, maize, and germinated sorghum flours. The pH progressively decreased with fermentation time, while titratable acidity increased from 0.029 to 0.118 ml/ml. Crude protein content increased with fermentation (251.7-274.8 mg/g) as a result of a shift in the dry matter composition. In-vitro protein digestibility markedly increased (12 %) as a result of fermentation. Protein solubility curves were above 30% of which highest for both fermented (12 and 24 hours) and unfermented composite flours were at pH 12 (51.77-77.64%) and lowest at pH 4 (30.31-35.98%). SDS-PAGE showed that protein hydrolysis occurred during fermentation over 12 and 24 hours. Unfermented composite flour was potentially stable as food ingredient due to its pasting stability, but the fermented flour low viscosity potential was preferred in this study as more flour will be used during porridge making, hence giving a food with a high nutrient density.

scholarly journals Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method

2014 ◽  
Vol 3 ◽  
Author(s):  
Iris M. Kawauchi ◽  
Nilva K. Sakomura ◽  
Cristiana F. F. Pontieri ◽  
Aline Rebelato ◽  
Thaila C. Putarov ◽  
...  
Keyword(s):  
Crude Protein ◽  
Pearson Correlation ◽  
Protein Solubility ◽  
Correlation Coefficients ◽  
Basal Diet ◽  
Protein Digestibility ◽  
Large Variability ◽  
The Relationship

AbstractAnimal by-product meals have large variability in crude protein (CP) content and digestibility. In vivo digestibility procedures are precise but laborious, and in vitro methods could be an alternative to evaluate and classify these ingredients. The present study reports prediction equations to estimate the CP digestibility of meat and bone meal (MBM) and poultry by-product meal (PM) using the protein solubility in pepsin method (PSP). Total tract CP digestibility of eight MBM and eight PM samples was determined in dogs by the substitution method. A basal diet was formulated for dog maintenance, and sixteen diets were produced by mixing 70 % of the basal diet and 30 % of each tested meal. Six dogs per diet were used to determine ingredient digestibility. In addition, PSP of the MBM and PM samples was determined using three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The CP content of MBM and PM ranged from 39 to 46 % and 57 to 69 %, respectively, and their mean CP digestibility by dogs was 76 (2·4) and 85 (2·6) %, respectively. The pepsin concentration with higher Pearson correlation coefficients with the in vivo results were 0·0002 % for MBM (r 0·380; P = 0·008) and 0·02 % for PM (r 0·482; P = 0·005). The relationship between the in vivo and in vitro results was better explained by the following equations: CP digestibility of MBM = 61·7 + 0·2644 × PSP at 0·0002 % (P = 0·008; R2 0·126); and CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P = 0·005; R2 0·216). Although significant, the coefficients of determination were low, indicating that the models were weak and need to be used with caution.

scholarly journals In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

2011 ◽  
Vol 11 (1) ◽  
pp. 147 ◽  
Author(s):  
Srilatha Kuntumalla ◽  
Quanshun Zhang ◽  
John C Braisted ◽  
Robert D Fleischmann ◽  
Scott N Peterson ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Shigella Dysenteriae ◽  
Protein Abundance ◽  
Shigella Dysenteriae Type ◽  
Vitro Protein
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