Protein Extraction Methods for Two-Dimensional Electrophoresis from Baphicacanthus cusia(Nees)Bremek Leaves-A Medicinal Plant with High Contents of Interfering Compounds

Protein extraction is a critical step for two-dimensional electrophoresis （2-DE）. Different plant samples require different and adaptive protein extraction protocols. The leaves of medicinal plant, Baphicacanthus cusia （Nees） Bremek are notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds （especially the secondary metabolites and pigments）. This study was aimed to establish a routine procedure for the proteomic analysis ofB. cusia leaves, and a new protocol for the protein extraction was developed by optimizing trichloroacetic acid （TCA）/ acetone extraction method. The efficiency of this protocol was demonstrated by comparison with 3 published protein extraction methods （chloroform/acetone, Mg/NP-40, Tris-base/acetone）. The results showed that the optimized TCA/ acetone precipitation extraction method gave a relatively high protein yield （9.263 mg g^-1 fresh weight）, high-resolution separation, clear protein profiles, the highest proteins spots （1 31 t protein spots）, and displayed less contamination in 2- DE gels. Therefore, the results suggested that the optimized TCA/acetone method was the most effective among the 4 methods for B. cusia leaves.

A Comparative Study of Soluble Protein Extractions of Populus deltoides × （ Trichocarpa × Deltoides） for 2-DE

Background： The disclosure of the poplar genome strengthens its position as well-established model organism. Populus has been subject of several proteome studies, but up to date no comparative study was performed on the extraction method of soluble proteins for this species. The extraction is the most critical step in two-dimensional gel electrophoresis and each extraction method has its advantages, disadvantages and limitations. Therefore protein extraction methods should be optimized for each tissue before starting an experimental setup. In prospect of future DIGE （Differential Gel electrophoresis） experiments for the investigation of the effects of cadmium and inoculation with plant growth promoting bacteria at the proteome level, the aim of this study was to optimize an extraction method for soluble proteins of poplar leaves and roots. Results： The acetone-phenol extraction method was found to be the most suited, rendering a high spot number and low background interference. During further optimization, several critical steps in the extraction method were revealed. Conclusion： Aiming to optimize the extraction of soluble leaf and root proteins of Populus deltoides × （trichocarpa× deltoides） compatible with DIGE analysis, a protocol rendering high reproducibility, low background interference and a high spot number was established, however no novel insights were acquired.

A robust protein extraction method for two dimensional electrophoresis of silkworm proteins

Silkworm (Bombyx mori) is a species of agricultural importance, as well as a model organism for Lepidoptera insects. Proteomic method has been widely used in silkworm research, and a robust mass spectrometry-compatible protein extraction method is urgently needed. In this study, we adapted phenol extraction method to extract silkworm midgut protein, and coupled this method with pH 5 - 8 gel strip for two dimensional electrophoresis. The phenol extraction method significantly increased the resolution, as well as greatly reduced the background of two dimensional electrophoresis gels. In addition, this method was well compatible with mass spectrometry analysis. This is the first report that phenol extraction method is used for silkworm midgut protein extraction, and may be applied in other researches.

Survey of Intracellular Protein Extraction Methods from Pichia pastoris

The broken efficiency of cell wall and the release amount of Pichia pastoris intracellular protein under different cell breaking conditions were investigated in this paper. The results showed that broken efficiency using hot alkali combined with high-pressure homogenizing method was higher than that of enzyme hydrolysis, hot alkali treatment and high-pressure homogenation, respectively. Suspended medium had little effect on the broken efficiency of yeast cell, but had significant effect on the protein release yield. The results indicated that optimal condition for intracellular proteins extraction was 30% (wet weight, w/v) of yeast cells suspend in 50 mM phosphate buffer (pH 10.0), water bathed at 60?C for 2 hours, homogenized twice at 100 MPa pressure. The broken efficiency of Pichia pastoris cell could reach 87.6% and the protein yield was 35.48 g per 100 g cells.

Mass Spectrometry-based Deep Coverage Proteome:Evaluation of Cellular Protein Extraction Methods

The current study comprehensively evaluates four different protein extraction methods based on urea,sodium dodecyl sulfate(SDS),anionic surfactants(BT),and total RNA extractor(Trizol),aiming to optimize the sample preparation workflow for mass spectrometry-based proteomics.Using HeLa cells as an example,we found that the method employing the mass spectrometry-compatible surfactant BT reagent significantly reduces the total time consumed for protein extraction and minimizes protein losses during the sample preparation process.Further integrating the four protein extraction methods,we identified over 7000 proteins from HeLa cells without relying on pre-fractionation techniques,and 2990 of them were quantified using label-free quantification.It is worth noting that the BT and SDS methods demonstrate higher efficiency in extracting membrane proteins,while the Urea and Trizol methods are more effective in extracting proteins from nuclear and cytoplasmic fractions.In summary,this study provides a novel solution for deep proteome coverage,particularly in the context of cellular protein extraction,by integrating mass spectrometry-compatible surfactants with traditional extraction methods to effectively enhance protein identification numbers.

Gut microbiota remodeling drived by dietary millet protein prevents the metabolic syndrome

Metabolic syndrome(Met S)is a chronic disease associated with the disturbance of gut microbiota homeostasis.Metabolites derived from gut microbes play essential roles in Met S prevention and therapy.Here,we focused on the inhibitory effect of the extract of millet bran protein(EMBP)on a high-fat diet(HFD)-induced Met S,aiming to identify gut microbiota and their metabolites that involve in the anti-Met S activity of EMBP.The obesity,chronic inflammation,insulin resistance in Met S mouse models were abolished after EMBP treatment.The protective mechanism of EMBP against HFD-induced Met S may depend on improved gut barrier function.Using microbiome analysis,we found that EMBP supplementation improved gut microbiome dysbiosis in Met S mice,specifically upregulating Bacteroides acidifaciens.The fecal microbiota transplantation(FMT)also demonstrated this phenomenon.In addition,metabolomic analysis showed that EMBP mediates metabolic profiling reprogramming in Met S mice.Notably,a microbiota-derived metabolite,gamma-aminobutyric acid(GABA),is enriched by EMBP.In addition,exogenous GABA treatment produced a similar protective effect to EMBP by improving NRF2-dependent gut barrier function to protect HFDinduced Met S.The results suggest that EMBP suppress host Met S by remodeling of gut microbiota as an effective candidate for next-generation medicine food dual purpose dietary supplement to intervene in MetS.

Recent Advances in Protein Extraction and Chiral Separation of Biomolecules

Reverse micelles create unique environment in organic media. They are capable of solubilizing hydrophilic biomolecules （e.g., proteins, peptides, amino acids, and DNAs） in their aqueous interior. This feature brings about the practical use of biomaterials in organic media because reverse micelles solubilize them with the intrinsic activity. In this paper, we focus on recent two topics concerning protein extraction and chiral separation of biomolecules using liquid membranes. In the first topic, we present recent attempts to extract proteins from an aqueous solution into isooctane using reverse micelles, and some important operational parameters to achieve an efficient protein transfer are discussed. Furthermore, novel function of reverse micelles as a protein activation medium is introduced. In the reverse micellar phase, denatured proteins were completely reactivated in the reverse micellar solution. The reverse micellar technique is found to be a useful tool not only for protein separation but also for protein refolding. Furthermore, we found that a cyclic ligand carixarene has an extraction ability to set up optimum conditions for protein transfer. In the second topic, we have found that a supported liquid membrane （SLM） encapsulating enzymes shows high enantioselectivity （enantioselective excess value is over 96%） in the transport of racemic pharmaceutical compound ibuprofen. A different experiment also suggests that the α-chymotrypsin-catalyzed reactions droved the enantioselective transport of L-phenylalanine based on the enantioselectivity of the enzyme. The SLM encapsulating the surfactant-enzyme complex enabled the highly enantioselective separation of racemic mixtures. It can be envisioned that arrangement of appropriate enzymes in the SLM system will allow enantioselective separation of various useful organic compounds.

Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal

The aim of this study was to optimize the conditions for the extraction of low-abundance proteins（LAPs） and the removal of abundant proteins（APs; β-conglycinin and glycinin） from soybean meal.Single factor and orthogonal experiments were designed to determine the effects of four factors（isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time） on protein concentration in isopropanol extracts.Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and matrix-assisted laser desorption/ionization-time of flight mass spectrometry（MALDI-TOF-MS）.The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h.Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L.Many LAPs were detected, including β-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins.The soybean APs（β-conglycinin and glycinin） were not found.The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs.

Deep eutectic solvents and alkaline extraction of protein from seabuckthorn seed meal: a comparison study

Seabuckthorn seed meal(SSM) is a waste of oil extraction industry that rich in protein. In order to seek suitable protein extraction method, three different deep eutectic solvents(DESs)(including choline chlorideglycerol, choline chloride-oxalic acid and choline chloride-urea) were developed for extracting protein from SSM and compared with alkaline. Result indicated that alkaline could effectively extract 56.9% protein from SSM and its protein content was 73.1%, higher than DES at 31.0%-41.4% and 64.3%-67.5%, respectively. However, compared to alkali, DES led to a product with less β-sheet, more β-turn, more essential amino acids, higher total amino acid content, especially choline chloride-urea which extracted protein showing an integrated and similar protein weight distribution compared to SSM. Also, this protein extracted chloride-urea showed a highest digestibility in vitro(by pepsin)(54.2%). These results indicated that choline chloride-urea extraction is better than alkaline extraction for SSM.

Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft

The precise mechanism of bone regeneration in different bone graft substitutes has been well studied in recent researches. However, miRNAs regulation of the bone formation has been always mysterious. We developed the anterior lumbar interbody fusion （ALIF） model in pigs using equine bone protein extract （BPE）, recombinant human bone morphogenetic protein-2 （rhBMP-2） on an absorbable collagen sponge （ACS）, and autograft as bone graft substitute, respectively. The miRNA and gene expression profiles of different bone graft materials were examined using microarray technology and data analysis, including self-organizing maps, KEGG pathway and Biological process GO analyses. We then jointly analyzed miRNA and mRNA profiles of the bone fusion tissue at different time points respectively. Results showed that miRNAs, including let-7, miR-129, m iR-21, miR-133, miR-140, miR-146, miR-184, and miR-224, were involved in the regulation of the immune and inflammation response, which provided suitable inflammatory microenvironment for bone formation. At late stage, several miRNAs directly regulate SMAD4, Estrogen receptor 1 and 5-hydroxytryptamine （serotonin） receptor 2C for bone formation. It can be concluded that miRNAs play important roles in balancing the inflammation and bone formation.

Antibacterial Activity of Bacillus subtilis and Properties of Protein Crude Extract

In order to get biological drugs with no resistance or toxic side effects and to reduce the use of antibiotics, a strain of Baci//us subtilis was isolated from animal intestine, and the isolate was identified by molecular biological method; in vitro an- tibacterial test of the isolate was performed using agar diffusion method; the optimal fermentation condition of the isoJate was screened by conventional culture method; the antibacterial crude protein of the isolate was extracted by saturated ammonium sulfate method; the physicochemical properties of antibacterial crude protein was de- tected by comparison method; The results showed that the isolate was B. subti/is, which had antibacterial effects on Staphy/ococcus aureus, streptococcus and swine erysipelas. The fermentation effect of the isolate was the best under the condition of temperature 30 ~C, pH 7, liquid volume 75 ml/250 ml, inoculation volume 20% and culture time 48 h. The antibacterial effect of the isolate was the best when extract- ed by 80% saturated ammonium sulfate. The antibacterial crude protein had strong resistance to heat and acid. Organic solvent and UV irradiation had some influences on antibacterial crude protein. Proteases had hydrolytic effects on antibacterial crude protein. The isolated B. subti/is can be used to prevent and control the diseases caused by S. aureus, streptococcus and swine erysipelas, and can regulate intesti- nal microecology by adding into expanded feeds.

An overview on bone protein extract as the new generation of demineralized bone matrix

Bone protein extract is regarded as the new generation of demineralized bone matrix. The aim of this paper is to describe and characterize the properties of demineralized bone matrix and its new generation product in addition to its application in animal and human studies. Bone protein extract has features of osteoconductivity, osteoinductivity and osteogenicity, which originate from its unique and precise processing. It has exhibited powerful bone formation capacity both in animal experiments and in clinical trials by providing an optimal microenvironment for osteogenesis. Furthermore, not only does it have excellent bio- compatibility, it also has good compatibility with other implant materials, helping it bridge the host and implanted materials. Bone protein extract could be a promising alternative for demineralized bone matrix as a bone graft substitute.

Oral immune regulation using colitis extracted proteins for treatment of Crohn's disease: Results of a phase Ⅰ clinical trial

AIM: To evaluate safety and possible efficacy of induction of oral immune regulation using colitis extracted proteins (CEP) in Crohn's disease (CD) subjects. METHODS: Ten CDs were treated orally with autologous CEP thrice weekly for 16 wk. Subjects were monitored for CDAI and IBDQ. Immune modulatory effect was assessed by T-lymphocyte FACS analysis, CEP-specific IFNγ ELISPOT assay and cytokine levels. RESULTS: Induction of oral immune regulation significantly ameliorated disease activity. All (10/10) subjects had clinical response (CDAI≤70) and 7/10 achieved clinical remission (CDAI≤150). Significant increase in mean IBDQ score was noted (134±9 vs 164±12). No treatment-related adverse events were noted. High levels of CEP-specific IFNγ spot forming colonies were detected in five subjects prior to treatment and in all five, a marked decrease was observed. The CD4+/CD8+ lymphocyte ratio and peripheral NKT cell numbers increased significantly, in 7/10 and in 5/10 subjects, respectively. Significant increase in serum IL-10 and IL-4 levels was observed in 7/10 subjects during treatment period. CONCLUSION: Immune regulation via oral administration of CEP is a safe and possibly effective treatment for subjects with moderate CD and may provide means of antigen-specific immune modulation.

Optimization of Protein Extraction and Decoloration Conditions for Tea Residues

To optimize alkaline method for extracting proteins from tea residue(TR), the effect of extraction conditions on tea protein extraction rate(TPER) was investigated. Single factor experiment showed the extraction temperature 80 °C, extraction time 100 min, p H value 13 and liquid–solid ratio 40:1 as the optimal extraction conditions. The orthogonal test revealed that the maximum TPER reached 29.71% under the following optimal combination of conditions: extraction temperature 70 °C, extraction time 60 min, p H 12 and liquid–solid ratio 50:1. For optimizing the purification of tea residue proteins, isoelectric point precipitation(p I), ammonium sulfate precipitation(a S) and isoelectric point plus ammonium sulfate precipitation(i PAS) were compared. The result showed that the highest protein precipitation rate(PPR) was 89.70% which was generated by using i PAS. Furthermore, powdered activated carbon was chosen as the most suitable decolorant for the extracted proteins.

Effect of Cigarette Smoke Extract on the Role of Protein Kinase C in the Proliferation of Passively Sensitized Human Airway Smooth Muscle Cells

To investigate the effect of cigarette smoke extract (CSE) on the role of protein kinase C (PKC) in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). After synchronization of cultured HASMCs, they were divided into a group A and Group B. The group A was treated with normal human serum and served as controls and the group B was treated with the serum of asthma patients. The group A was further divided into group of A_1, A_2 and A_3 and the group B was sub-divided into the group of B_1, B_2, B_3, B_4 and B_5. No other agents were added to the group A_1 and B_1. The cells of group A_2 and B_2 were stimulated with 5 % CSE for 24 h. HASMCs from group A_3 and B_3 were treated with PKC agonist PMA (10 nmol/L) and CSE (5 %) for 24 h. PKC inhibitor Ro-31-8220 (5 μmol/L) was added to the HASMCs of group B_4 for 24 h. The cells from group B_5 were stimulated with Ro-31-8220 (5 μmol/L) and CSE (5 %) for 24 h. The proliferation of HASMCs isolated from group A and B was examined by cell cycle analysis, MTT colorimetric assay and 3H-TdR incorporation test. The expression of PKC-α in each group was observed by Western blotting and RT-PCR, respectively. The results showed that the percentage of S phase, absorbance (A) value, the rate of 3H-TdR incorporation, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_1, B_2 and B_3 were significantly increased compared to those of group A_1, A_2 and A_3 correspondingly and respectively (P<0.01). The proliferation of HASMCs of group A_2 and B_2 stimulated with CSE and group A_3 and B_3 stimulated with CSE and PMA were also significantly enhanced when group A_1, A_2 and A_3 and group B_1, B_2 and B_3 compared to each other (P<0.05, P<0.01, respectively). The percentage of S phase, absorbency (A) value, 3H-TdR incorporation rate, the ratios of A value of PKC-α mRNA and the A value of PKC-α protein in HASMCs from group B_4 treated with Ro-31-8220 and group B_5 treated with CSE and Ro-31-8220 were significantly decreased as compared to those of group B_1 and B_2 correspondingly and respectively (P<0.05, P<0.01). It was concluded that CSE can enhance the passively sensitized HASMC proliferation and the expression of PKC alpha. PKC and its alpha subtype may contribute to this process. Our results suggest cigarette may play an important role in ASMCs proliferation of asthma through PKC signal pathway.

Research Progress of Value, Extraction and Applicationof Silkworm Chrysalis Protein

Silkworm chrysalis protein is a high quality pure natural complete animal protein with high utilization value, and it has been widely usedin many industries in recent years. The nutrition, health care and medicinal value of silkworm chrysalis protein, the degreasing, deodorization anddecoloration extraction process, and its application in food, medicine, feed, etc. are summarized, in order to provide the reference for further development and industrial production of silkworm chrysalis protein.

A Cooperative Approach for the Extraction of Protein Motifs

By integrating the concept of cooperative approach, an extension of the fast annealing coevolutionary algorithm is presented in this paper. It outperformed the original algorithm in the domain of function optimization, especially in terms of convergence rate. It was also applied to a real optimization problem, protein motif extraction. And a satisfactory result has been obtained with the accuracy of prediction achieving 67.0%, which is in agreement with the result in the PROSITE database.

Comparison of Protein and Amino Acids in the Extracts of Two Edible Mushroom, <i>Pleurotus sajor-caju</i>and <i>Schizophyllum commune</i>

This study was undertaken to determine total protein (%) and profiles of amino acid and made comparison between the aqueous and organic solvent extracted mushroom. Extraction was made from two edible, Pleurotus sajor-caju (commercial) and Schizophyllum commune (wild) types of mushrooms. Four types of solvents were used for the extraction include 100% aqueous, 50% ethanol, 50% methanol and 50% acetone. True protein of mushroom extract was analyzed with colorimetric Lowry method and amino acids were determined by using high-performance liquid chromatograph (HPLC). The range of 1.06% to 3.43% and 1.30% to 2.17% total protein value were obtained in the extracts of P. sajor-caju and S. commune respectively, while the highest total protein of 3.43% was determined in aqueous extracted P. sajor-caju mushroom. The amount of total amino acids of S. commune and P. sajor-caju were in the range of 308.65 mg/g to 443.84 mg/g and 172.52 mg/g to 400.76 mg/g, respectively. The highest content of 443.84 mg/g total amino acids and 77.08 mg/g of essential amino acids were obtained in the aqueous extracted Schizophyllum commune. On the other hand the total content of essential amino acids (EAA). Essential amino acid of both mushrooms was dominated by leucine along with threonine and alanine, but the highest contents were determined from the extract of Schizophyllum commune. Aqueous extraction was effective in both types of mushroom for the protein components as well essential amino acids compared to other organic solvents that were used in extraction process in this study.

Extraction and identification of membrane proteins from black widow spider eggs

The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider （Latrodectus tredecimguttatus）, and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCI buffer containing 4% CHAPS and 2% DTT （pH 7.4）. SDS-PAGE combined with nLC- MS/MS identified 39 proteins with membranelocalization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

Size Changes of Reverse Micelles after Extraction of Peanut Protein and Their Forward Extraction Rates

The aim of this study was to detect the size changes of reverse micelles after extraction of peanut protein and their forward extraction rates. Factors that affect the size of reverse micelles and the extraction of peanut protein were also investigated. The size of reverse micelles and the size changes were measured according to the theory of dynamic light scattering under different conditions such as different sodium bis(2-ethylhexyl) sulfosuccinate(AOT) concentrations, p H values, ion concentrations, and salt species.With the increase of AOT surfactant concentrations in a certain range, the size of empty and full reverse micelles increased and the forward extraction rate decreased. The effect of pH on empty reverse micelles was not significant. However, the effect of pH on the full reverse micelle size and forward extraction rate were significant. Its forward extraction rate increased to the maximum39.6% at pH 7.5. The increase of the salt concentration of a buffer solution in a certain range decreased the size of empty and full reverse micelles and reduced the forward extraction rate of peanut protein. Ionic species had important effects on reverse micelles and peanut protein extraction. An increase in the amount of buffer solution enlarged the empty reverse micelle size in 0.03%-0.11%(V/V). However, it did not translate to a larger reverse micelle size. The size of the empty reverse micelles containing K_2SO_2 reached 24.1 nm with a 0.19%(V/V) buffer solution added. The sizes of the full reverse micelles were larger than those of the empty reverse micelles after forward extraction. However, maximum sizes were achieved with the addition of a 0.03%(V/V) buffer solution. The amount of 0.03%(V/V) buffer solution added was appropriate for extracting peanut protein.