Crude Protein Mixtures Research Articles

Efficacy of a phytonutrient supplement at different dietary protein contents on growth performance, gut morphology, digestive enzymes activities and stress indicators of broilers subjected to circular heat stress

Context Dietary manipulations such as reductions in protein or the addition of bioactive feed additives might alleviate the adverse effects of high ambient temperature in poultry. Aims The effects of dietary crude protein (CP) and phytonutrient mixture (PM) on growth performance and physiological responses were evaluated in broilers under circular heat stress (HS). Methods In total, 420 1-day-old broiler chickens were randomly distributed to seven treatments in a factorial arrangement (3 × 2), with three levels of CP (normal, medium (95% of the normal CP content) and low (90% of the normal CP content)) and with or without the addition of PM (mixture of 5 mg/kg carvacrol, 3 mg/kg cinnamaldehyde, and 2 mg/kg capsicum oleoresin). The thermoneutral control (TNC) birds were housed in a thermoneutral chamber and fed with a normal-CP diet without PM supplementation. The other six groups were kept in a HS chamber (33°C) for 8 h (1000 hours to 1800 hours). Key results After 6 weeks, feeding broiler chickens with low-CP diets had adversely affected growth performance and intestinal protease activities, while mortality rate, heterophile to lymphocyte ratio, and serum cortisol concentration of the low-CP group were lower (P < 0.05) than those of the high-CP group. Dietary PM supplementation also positively influenced (P < 0.05) average daily gain, feed conversion ratio, intestinal lipase and trypsin activities and serum T4 concentration. There were the CP × PM interactions (P < 0.05) for jejunal villus height and villus surface area, indicating that the effect of PM on these responses was more marked at the lower dietary CP contents. Conclusions Supplementation with PM can ameliorate the detrimental effects of HS on productive performance and digestive enzyme activities in broilers. The PM supplementation could improve intestinal morphology when applied to the diets of heat-stressed broilers with lower CP contents (95% and 90% of the recommendations). Implications Although growth performance was reduced by feeding low-CP diets (90% of the normal CP content), a beneficial effect was observed on the stress indicators of broilers grown in HS conditions. Dietary supplementation of PM might also be an effective strategy to alleviate the adverse effect of HS.

Effect of Key Parameters on Jacalin Extraction from Aqueous Phase into Anionic Reverse Micelles

This study demonstrates the extraction of jacalin from crude extract of jackfruit seeds using anionic surfactant based reverse micellar system, sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. Effects of various parameters, such as, NaCl concentration (0.05 – 1 M), pH of aqueous phase (pH 4 – 10), AOT concentration (5 – 150 mM), contact time (5 – 30 min) and phase volume ratio (0.5-5) on the transfer efficiency of jacalin was evaluated by changing one factor at a time (OFAT) while keeping the other parameters constant. A maximum of 83% of protein transfer was achieved after equal volume of organic and aqueous phase was stirred for 20 min using 20 mM AOT at pH 5 aqueous phase containing 0.1 M NaCl. The findings demonstrated AOT reverse micellar system as a promising and effective method to extract and purify jacalin from crude protein mixture.

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica.

Mesoporous silica materials are promising carriers for enzyme immobilization in heterogeneous biocatalysis applications. By tailoring their pore structural framework, these materials are designable for appropriate enzyme binding capacity and internal diffusivity. To supply O2 efficiently to solid-supported immobilized enzymes represents a core problem of heterogeneously catalyzed oxidative biotransformations. In this study, therefore, we synthesized and compared three internally well-ordered and two amorphous silica materials as enzyme carriers, each of those with pore sizes of ≥10 nm, to enable the coimmobilization of d-amino-acid oxidase (79 kDa) and catalase (217 kDa). Both enzymes were fused to the silica-binding module Zbasic2 to facilitate their selective and oriented immobilization directly from crude protein mixtures on native silica materials. Analyzing the effects of varied pore architecture and internal surface area on the performance of the immobilized bienzymatic system, we showed that a uniform pore structural framework was beneficial for enzyme loading (≥70 mg protein/g carrier), immobilization yield (≥90%), surface and pore volume filling without hindered adsorption, and catalytic effectiveness (≥60%) of the coimmobilizate. Using the best carrier LP-SBA-15, we obtained a solid oxidase-catalase preparation with an activity of 2000 μmol/(min g_material) that was recyclable and stable during oxidation of d-methionine. These results affirm a strategy of optimizing immobilized O2-dependent enzymes via tunable internal structuring of the silica material used as carrier. They thus make a significant advance toward the molecular design of heterogeneous oxidation biocatalysts on mesoporous silica supports.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Western Blotting Using PVDF Membranes and Its Downstream Applications.

Western blotting using polyvinylidene difluoride (PVDF) membranes is one of the most popular techniques for detection and characterization of proteins. If this technique is combined with immunodetection, the behavior of a particular protein can be clarified. On the other hand, if it is combined with Edman sequencing, the primary structure of the protein can be determined. A protein sample is transferred from an SDS-polyacrylamide gel electrophoresis (PAGE) gel onto a PVDF membrane by electroblotting. The membrane carrying the protein is either used for immunodetection or protein sequencing. SDS-PAGE followed by Western blotting combined with immunodetection using antibodies can easily detect protein behavior in crude protein mixtures. Furthermore, two-dimensional PAGE followed by Western blotting and Edman sequencing allows effective sequence determination of crude protein mixtures that may not be easily purified by conventional column chromatography.

Multi‐dimensional fractionation and characterization of crude protein mixtures: Toward establishment of a database of protein purification process development parameters

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.

Isolation and Characterization of Novel Tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.

Omics tools in accelerated purification process development: Multidimensional fractionation and characterization of crude protein mixtures

Until recently, process development was restricted to a sequential experience based regime. However, new innovativemethods have been developed to perform e.g. up to 96 parallel chromatographic experiments on a Tecan Freedom EVO® robotic workstation, allowing for thermodynamic, kinetic, dynamic binding, and elution studies in an automated, parallelized, and miniaturized mode, which saves time, money, and target molecule. In the era of accelerated time lines to launch new products on the market, traditional experience driven process development seems long outdated. While other fields are front runner in using modern technologies, process development labs in the biotechnological industry are only approaching high throughput techniques, mathematical tools or the computational power available for a few years. Modern high throughput platforms allow the miniaturisation and automation of nearly every unit operation used in biopharmaceutical production. Next to experimental tools for solubility studies, aqueous extractions or IB refolding we will present the complete range of tools for chromatographic process development. Using for example miniaturized chromatographic columns in a 96 well format it becomes possible to either screen chromatographic procedures 8 fold in parallel or to mimic complete multi step chromatographic procedures on an automated HTS platform. In addition the necessity of intelligent screening approaches using mathematical searchalgorithmsand the appropriate high end analytical approaches will be discussed.With such a tool in hand, issues arising in process development can be answered with a limited amount of feedstock and manpower. Case studies will be given using model proteins but also therapeutic targets such as Insulin, mAb and pDNA.

Kinetic Binding Analysis of Aptamers Targeting HIV-1 Proteins by a Combination of a Microbalance Array and Mass Spectrometry (MAMS)

An enhanced chip-based detection platform was developed by integrating a surface acoustic wave biosensor of the Love-wave type with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The system was applied to characterize the interaction of aptamers with their cognate HIV-1 proteins. The aptamers, which target two proteins of HIV-1, were identified using an automated in vitro selection platform. For aptamers S66A-C6 and S68B-C5, which target the V3 loop of the HIV-1 envelope protein gp120, KD values of 406 and 791 nM, respectively, were measured. Aptamer S69A-C15 was shown to bind HIV-1 reverse transcriptase (HIV-1 RT) with a KD value of 637 nM when immobilized on the biosensor surface. HIV-1 RT was identified with high significance using MALDI-ToF MS even in crude protein mixtures. The V3-loop of gp120 could be directly identified when using chip-bound purified protein samples. From crude protein mixtures, it could be identified indirectly with high significance via its fusion-partner glutathione-S-transferase (GST). Our data show that the combination of the selectivity of aptamers with a sensitive detection by MS enables the reliable and quantitative analysis of kinetic binding events of protein solutions in real time.

Protein nanofibres of defined morphology prepared from mixtures of crude crystallins

Self assembling protein nanofibres have great potential within the nanotechnology industry. To realise this potential, nanofibres need to be manufactured on a commercial scale. To date, amyloid fibrils, a form of protein nanofibre with desirable bionanotechnological properties, have only been produced using expensive and time-costly, small-scale methods. This paper describes the production of protein nanofibres from semi-pure and crude mixtures of bovine crystallin proteins, currently a waste product in the meat industry. These nanofibres displayed amyloid fibril characteristics. Moreover, distinct fibre morphologies were observed dependent upon the formation conditions adopted. This research shows that amyloid fibrils can be inexpensively produced from crude crystallin protein mixtures, making them a potential industrial source for protein nanofibres.

Effects of Supplementation and Stage of Lactation on Performance of Grazing Dairy Ewes

The majority of dairy sheep in the world are fed pasture and supplemental grain during lactation; however, no trials have reported the effects of supplementation of dairy ewes grazing improved pastures in North America. In trial 1, 56 three-year-old grazing dairy ewes in early [21±10 d in milk (DIM)] or late (136±9 DIM) lactation were fed 0 or 0.82kg of dry matter/d per ewe of supplement (16.5% crude protein mixture of corn and a soybean meal-based high-protein pellet) in a 2×2 factorial arrangement of treatments. There were no significant interactions between stage of lactation and supplementation treatments. Average test-day milk production was higher in early-lactation ewes than in late-lactation ewes (1.74 vs. 1.21kg/d, respectively). Although test-day milk protein percentage was higher in late-lactation ewes than in early-lactation ewes (5.02 vs. 4.86%, respectively), there was no difference in milk fat percentage between stages of lactation. Supplemented ewes had higher milk production (1.59 vs. 1.36kg/d, respectively), lower milk fat percentage (5.75 vs. 6.00%, respectively), and lower milk protein percentage (4.84 vs. 5.04%, respectively) than unsupplemented ewes. Milk urea N levels were similar between the 2 stages of lactation and between the 2 supplementation treatments and were above recommended levels for dairy sheep, indicating an excess intake or inefficient utilization of protein for both supplementation treatments. In trial 2, 96 two-, three-, and four-year-old grazing dairy ewes in midlactation (112±21 DIM) were randomly assigned to 4 treatments of 0, 0.41, 0.82, or 1.24kg of dry matter/d per ewe of whole corn. Average test-day milk production increased linearly and milk fat percentage decreased quadratically with increasing amounts of corn supplementation. Milk protein yield increased linearly, and milk urea N levels decreased quadratically with increasing amounts of corn supplementation, suggesting an improvement in the utilization of pasture protein with increasing dietary energy intake.

PH-gradient ion-exchange chromatography: An analytical tool for design and optimization of protein separations

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (p I < 6) and basic (p I > 8) proteins elute roughly at their p I, whereas neutral proteins (p I 6–8) elute at pH 8–9 regardless their p I values. Because of the flat nature of protein titration curves from pH ∼6 to ∼9, neutral proteins indeed exhibit nearly zero net charge at pH ∼9. The elution-pH in pH-gradient IEC or the titration curve, but not the p I, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.

Leucyl/phenylalanyl(L/F)‐tRNA‐protein transferase‐mediated aminoacyl transfer of a nonnatural amino acid to the N‐terminus of peptides and proteins and subsequent functionalization by bioorthogonal reactions

We report here a new strategy for derivatizing peptides and proteins at the N-terminus. To achieve this, a nonnatural amino acid was charged onto a tRNA and then enzymatically transferred to a lysine (Lys) unit at the N-terminus of a peptide or a protein by using L/F-tRNA-protein transferase. By using the chemoenzymatic technique, beta-(2-quinolyl)-L-alanine, p-azido-L-phenylalanine, and p-acetyl-L-phenylalanine were introduced to the N-terminus. The latter two nonnatural amino acids possess bioorthogonal functional groups to which artificial tags can be introduced. Actually, a biotin tag was coupled to the bioorthogonal ketone group of acetylphenylalanine at the N-terminus of a peptide. N-terminal-specific biotinylation and fluorescence derivatization of the bioorthogonal azido-containing protein or peptide was also carried out based on a [3 + 2] cycloaddition. The enzymatic transfer of a nonnatural amino acid to the N-terminus of target peptides or proteins was also successfully achieved in the presence of other peptides or crude protein mixtures.

A Conserved Subtilisin Protease Identified in Babesia divergens Merozoites

Invasion of erythrocytes is an integral part of the Babesia divergens life cycle. Serine proteases have been shown to play an important role in invasion by related Apicomplexan parasites such as the malaria parasite Plasmodium falciparum. Here we demonstrate the presence of two dominant serine proteases in asexual B. divergens using a biotinylated fluorophosphonate probe. One of these active serine proteases (p48) and its precursors were recognized by anti-PfSUB1 antibodies. These antibodies were used to clone the gene encoding a serine protease using a B. divergens cDNA library. BdSub-1 is a single copy gene with no introns. The deduced gene product (BdSUB-1) clearly belongs to the subtilisin superfamily and shows significant homology to Plasmodium subtilisins, with the highest degree of sequence identity around the four catalytic residues. Like subtilisin proteases in other Apicomplexan parasites, BdSUB-1 undergoes two steps of processing during activation in the secretory pathway being finally converted to an active form (p48). The mature protease is concentrated in merozoite dense granules, apical secretory organelles involved in erythrocyte invasion. Anti-PfSUB1 antibodies have a potent inhibitory effect on erythrocyte invasion by B. divergens merozoites in vitro. This report demonstrates conservation of the molecular machinery involved in erythrocyte invasion by these two Apicomplexan parasites and paves the way for a comparative analysis of other molecules that participate in this process in the two parasites.

Analysis of proteolytic degradation of a crude protein mixture using a surface acoustic wave sensor

Degradation of a crude protein mixture by proteases with pH optima from acidic to basic was followed in real time using a surface acoustic wave biosensor in Love-wave geometry. Proteases EC 3.4.23.18 from Aspergillus saitoi, EC 3.4.21.62 from Bacillus licheniformis, and Novozyme from Bacillus sp. have been used. Kinetic constants extracted from the curves resulted for comparable protease concentrations in maximal degradation rates between 1.1 × 10 −2 and 1.5 × 10 −2 s −1. For the three proteases investigated, similar amounts of up to about two-thirds of the estimated 28 ng/cm 2 bound molecules were proteolyzed. The residual material not degraded by the proteases was removed from the surface with 0.5% SDS. The analysis of the sensor signal allows: (1) estimation of the total mass of protein bound to the sensor surface and of the degradable fraction; (2) extraction of the pure mass signal; and (3) kinetic evaluation.

Chromatographic Conditions for the Analytical Separation of Crude Peptide and Protein Mixtures

Chromatographic Conditions for the Analytical Separation of Crude Peptide and Protein Mixtures

Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein–protein interactions or protein–small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.

Denaturing Isoelectric Focusing Free-Flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures

Native Isoelectric Focusing Free-flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures Peter J.A. Weber, Gerhard Weber, and Christoph Eckerskorn This protocol was adapted from "Native IEF-FFE Fractionation of Crude Protein Mixtures," contributed by Peter J.A. Weber, Gerhard Weber, and Christoph Eckerskorn, Chapter 19, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.

Native Isoelectric Focusing Free-flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures

Isoelectric Focusing Free-flow Electrophoresis (IEF-FFE) Fractionation of Crude Protein Mixtures Peter J.A. Weber, Gerhard Weber, and Christoph Eckerskorn This protocol was adapted from Native IEF-FFE Fractionation of Crude Protein Mixtures, contributed by Peter J.A. Weber, Gerhard Weber, and Christoph Eckerskorn, Chapter 19, in Purifying Proteins for Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2004.