Protein estimation in tissues containing high levels of lipid: modifications to Lowry's method of protein determination

A method to estimate protein in detergent-solubilized homogenates of lipid-rich biological samples (e.g., adipose tissue, myelin-enriched fractions of sheep brain) is described. The method is also suitable for samples in which protein is present as a protein-detergent complex. The method involves homogenization of tissue in the presence of a suitable detergent and KCl. Protein is then estimated in an aliquot of this homogenate by Lowry's method in the presence of excess sodium dodecyl sulfate, the solutions being clarified by extraction with ethyl acetate. Protein solubilization by Triton X-100 from adipose tissue was biphasic, extracting two to three times more protein under optimum conditions [1.7 +/- 0.1% (v/v) Triton X-100 and 0.75 M KCl], compared with homogenization without salt and detergent. Unlike adipose tissue, protein solubilization from myelin-enriched fractions of sheep brain peaked at 1% (v/v) Triton X-100, resulting in the extraction of approximately three times more protein than homogenization in the absence of detergent and salt.     

Solubilization of Collagen-Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.     

Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human myocardium

In order to maximize the myocardial proteome observed by two-dimensional gel electrophoresis (2-DE), the effect of (1) either an ionic or different zwitterionic detergents during tissue homogenization and (2) altering the "standard" detergent for isoelectric focusing (3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS)) to either the zwitterionic detergent amidosulfobetaine-14 (ASB-14) or N-decyl-N-N'-dimethyl-3-ammonio-1-propane sulfonate (SB3-10) was investigated. Sodium dodecyl sulfate was shown to be a superior detergent for extraction of proteins during homogenization of cardiac tissue compared to the detergents ASB-14, SB3-10 or CHAPS. Additionally, both ASB-14 and SB3-10 exhibited better extraction than CHAPS for distinct regions of two-dimensional gels. In most cases, the best combination of homogenization and focusing conditions did not involve the use of the same detergent. Specifically, it was found that the ability to mix homogenization and focusing conditions can allow one to obtain an optimum balance between the resolution and number of protein spots obtained in 2-DE analysis of cardiac tissue. An excellent initial combination of buffers to utilize for the general examination of cardiac proteins was determined to be initial homogenization in a buffer containing ASB-14 followed by focusing in a buffer containing CHAPS.     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

A high-yield method to extract peptides from rat brain tissue

A process to extract and enrich extracellular peptides and proteins from tissues should have broad utility in the burgeoning proteomics field. To address this need, a novel three-step protocol was developed to extract polypeptides from whole tissue samples and enrich the extracellular components. The initial homogenization of rat brain was carried out at neutral pH to optimize protein and peptide stability and solubility. Subsequent covalent chromatography on an activated thiopropyl resin was employed to debulk the tissue extract by selectively removing a substantial fraction of the intracellular protein component under nondenaturing conditions. Finally, extraction with 0.1% trifluoroacetic acid was used to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. The fractions from each step in the process were compared to a single extract obtained by homogenization in 0.5 M acetic acid. The recovery and yields of endogenous neuropeptides and an exogenously added peptide were evaluated by enzyme immunoassay and Western blotting, respectively. In summary, the three-step protocol was superior to the extraction of tissue with 0.5 M acetic acid in terms of peptide recovery, enrichment, and sample stability. Enrichment of the extracellular protein compartment from tissues should be valuable in proteomics experiments aimed at identifying biomarkers that can partition into serum.     

A method for the isolation of rat submandibular salivary gland polysomes on linear sucrose density gradients

A procedure for isolating rat submandibular salivary gland polysomes on sucrose density gradients has been described. Electron micrographs of the gland revealed the existence of a very well-developed system of membranebound ribosomes. These morphological findings dictated the use of vigorous homogenization of tissue, a detergent, and low g-forces during tissue fractionation procedures for maximal recovery of this cellular organelle. The methodology allowed for the isolation of polysomes susceptable to RNase degradation and able to incorporate labeled amino acids into protein in a cell-free system.     

Study of detergent-mediated liberation of hepatitis B virus-like particles from S. cerevisiae homogenate: identifying a framework for the design of future-generation lipoprotein vaccine processes

Virus-like particles (VLPs) are expressed intracellularly in Saccharomyces cerevisiae and the recovery process involves the use of a detergent, which facilitates the release of VLP from host cell components. The detergent-mediated liberation of VLPs is a critical step in primary recovery and is responsible for setting the backdrop for subsequent purification in terms of product yield and characteristics of the process stream. In this paper the use of Triton X-100 detergent for the recovery of lipid envelope VLPs, using the hepatitis B surface antigen (HBsAg) as the VLP model, was investigated. To develop a framework that can be adopted in process design for future generation VLP vaccine candidates, the impact of Triton X-100 was characterized via different response factors: (i) recovery and activity of the HBsAg; (ii) level of protein and lipid contamination from the host cell; and (iii) indirect impact on the performance of an ultrafiltration step following primary recovery. Our studies identified that an increase in detergent concentration favors recovery of HBsAg only to a specific threshold, 0.5% v/v Triton X-100. Further increase in detergent results in delipidation of HBsAg leading to loss in antigenic activity. The level of contamination due to host protein and lipid co-liberation is in proportion with the amount of detergent employed. Greater membrane resistance during ultrafiltration was observed for samples generated using higher concentrations of detergent due to the increase in membrane fouling by the contaminants. Based on this study, Triton X-100 concentrations in the range of 0.2-0.5% v/v appears to be most suitable for recovery of native HBsAg. Choosing between 0.2-0.5% v/v would involve identifying a suitable tradeoff between desired product yield and the level of contamination that can be tolerated by downstream operations.     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane     

Effects of stage of maturity on ensiling characteristics and ruminal nutrient degradability of oat silage

A study was conducted to determine the effects of stage of maturity on ensiling characteristics and ruminal nutrient degradability of oat silage. Oat was field grown and forage was harvested at the boot or soft dough stage and ensiled in mini-silos for 0, 2, 4, 8, 16 and 45 days. Two lactating Holstein cows fitted with ruminal fistulas were used determine ruminal nutrient degradability. Regardless of the stage of maturity, ensiled forages went through a rapid fermentation with a sharp decline in pH during the first 2 days of ensiling. Extensive proteolysis took place between 0 and 2 days as indicated by a reduction in true protein and neutral detergent insoluble protein (NDICP) and an increase in non-protein nitrogen (NPN). Chemical analysis of the 45 days silage showed that stage of maturity had no effect on neutral detergent fibre (NDF) and acid detergent fibre (ADF) of oat silage. However, oat harvested at the boot stage contained more crude protein (CP) and less starch than that harvested at the soft dough stage. Distribution of protein fractions showed that oat harvested at the boot stage contained lower NPN, NDICP and acid detergent insoluble protein than oat harvested at the soft dough stage. Results of the in situ incubation experiment indicated that oat harvested at the soft dough stage had lower ruminal dry matter (60.6 vs. 66.4%), CP (81.3 vs. 88.7%) and NDF (35.4 vs. 42.2%) degradabilities than oat harvested at the boot stage. It was concluded that chemical composition and ruminal nutrient degradability of oat silage are significantly influenced by stage of maturity.     

亲和介质及溶液条件对蛋白质溶液中内毒素去除的影响

生物制品中内毒素的去除是一项十分重要的工作。为了更好地去除各种生物制品中的内毒素,采用合成的多粘菌素B琼脂糖亲和介质,通过静态吸附的方法去除蛋白质溶液中的内毒素。重点考察了介质的间臂长度、配基密度以及各种溶液条件(pH值、盐种类和浓度、蛋白质种类和浓度、内毒素浓度、添加剂等)对内毒素去除率及蛋白质回收率的影响。分别采用动态浊度法和Lowry法检测内毒素含量和蛋白质浓度。结果表明该介质具有载量高、去除速度快、去除率高、可重复使用的特点。此外,配基密度、pH值、盐浓度和蛋白质特性(等电点和疏水性)对内毒素去除效果均有重要影响。在优化的条件下,血红蛋白、人血清白蛋白和溶菌酶的回收率分别达到87.2%、73.4%和97.3%,相应的内毒素去除率分别达到99.8%、97.9%和99.7%。阐明了各种因素对内毒素去除率和蛋白质回收率的影响规律,为生物制品中内毒素的高效去除提供了参考。     

Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid‐envelope virus‐like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving ～70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of ～80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions (～400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low‐shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two‐fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues

Hypoxia-inducible factor-1 alpha (HIF-1α) is an important marker of hypoxia in human tumors and has been implicated in tumor progression. Drugs targeting HIF-1α are being developed, but the ability to measure drug-induced changes in HIF-1α is limited by the lability of the protein in normoxia. Our goal was to devise methods for specimen collection and processing that preserve HIF-1α in solid tumor tissues and to develop and validate a two-site chemiluminescent quantitative enzyme-linked immunosorbent assay (ELISA) for HIF-1α. We tested various strategies for HIF-1α stabilization in solid tumors, including nitrogen gas-purged lysis buffer, the addition of proteasome inhibitors or the prolyl hydroxylase inhibitor 2-hydroxyglutarate, and bead homogenization. Degassing and the addition of 2-hydroxyglutarate to the collection buffer significantly increased HIF-1α recovery, whereas bead homogenization in sealed tubes improved HIF-1α recovery and reduced sample variability. Validation of the ELISA demonstrated intra- and inter-assay variability of less than 15% and accuracy of 99.8 ± 8.3% as assessed by spike recovery. Inter-laboratory reproducibility was also demonstrated (R² = 0.999). Careful sample handling techniques allow us to quantitatively detect HIF-1α in samples as small as 2.5 μg of total protein extract, and this method is currently being applied to analyze tumor biopsy specimens in early-phase clinical trials.     

Solubility of vesicular stomatitis virus M protein in the cytosol of infected cells or isolated from virions.

The peripheral membrane M protein of vesicular stomatitis virus purified by detergent extraction of virions and ion-exchange chromatography was determined to be a monomer in the absence of detergent at high salt concentrations. Reduction of the ionic strength below 0.2 M resulted in a rapid aggregation of M protein. This self-association was reversible by the detergent Triton X-100 even in low salt. However, aggregation was not reversible by high salt concentration alone. M protein is initially synthesized as a soluble protein in the cytosol of infected cells, thus raising the question of how the solubility of M protein is maintained at physiological ionic strength. Addition of radiolabeled M protein purified from virions to unlabeled cytosol from either infected or uninfected cells inhibited the self-association reaction. Cytosolic fractions from infected or uninfected cells were equally effective at preventing the self-association of M protein. Self-association could also be prevented by an irrelevant protein such as bovine serum albumin. Sedimentation velocity analysis indicated that most of the newly synthesized M protein is monomeric, suggesting that the solubility of M protein in the cytosol is maintained by either low-affinity interaction with macromolecules in the cytosol or interaction of a small population of M-protein molecules with cytosolic components.     

The purification of cell nuclei from calf uterus by zonal centrifugation in reorienting density gradients

A method is described for isolation of substantial amounts of pure and enzymatically active nuclei from whole calf uterus. The technique involves a multistep sequential homogenization of the tissue and a zonal centrifugation of the crude nuclear preparation in a reorienting density gradient rotor. Electron and phase contrast microscopic observations show that the nuclei are intact and practically free from cytoplasmic contamination. Based on DNA recovery, the purified fraction contains 9% of the nuclei of the total tissue and more than 19% of the filtered homogenate. The pure nuclear fraction consists of 29% DNA, 7% RNA, and 64% protein, which parallels the composition of purified nuclei from other mammalian tissues.     

Solubilization and characterization of yeast signal peptidase

An efficient post-translational assay for solubilized yeast signal peptidase has been developed. The enzyme can be solubilized in nonionic detergent (0.5% Nikkol) without added salt, but salt increased the efficiency of solubilization. Radiosequencing of the cleaved substrate revealed that the enzyme removed the signal peptide. The substrate (prepro-alpha-factor) must be pretreated with sodium dodecyl sulfate to be cleaved. The enzyme displays a broad, alkaline pH optimum, retaining activity at pH 12. Moderately high temperatures (35 degrees C), excess detergent (greater than 0.5% Nikkol), or high salt (greater than 300 mM KOAc) will inactivate the enzyme. Phosphatidylcholine is necessary for optimal activity. The optimal ratio of Nikkol:lipid:sodium dodecyl sulfate is 6.4:2.2:1. The membrane association of yeast signal peptidase is resistant to carbonate extraction, indicating that it is an integral membrane protein.     

Solubilization, partial purification and photolabeling of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT).

In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n-octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6-diphenyl-1,3,5-hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one-tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I-labelled-18-(4'-azido-2'-hydroxybenzoylamino)-oleyl-CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme.     

Expression, purification and biological properties of the carboxyl half part of the HTLV-I surface envelope glycoprotein

The carboxyl half of the surface envelope protein of HTLV-I contains the major immunodominant and neutralizable domains. Using two affinity chromatography steps and a combination of high salt concentration and non-ionic detergent, we purified this part of the envelope protein from Escherichia coli. Analysis of some immmunological and biological properties of this protein indicated that it was folded in a way that preserved the correct structure of this domain of the HTLV-I envelope protein. It could be utilized in structural studies to further understand the mechanisms of HTLV-I entry and to better define the component(s) of an effective vaccine.     

Overproduction and high-yield purification of phospholipase A from the outer membrane of Escherichia coli K-12

The cloned gene for the outer-membrane-bound phospholipase A from Escherichia coli was placed under control of the strong PL promoter of phage lambda. Induction of PL resulted in a 250-fold overexpression up to about 2% total cellular protein. This overproduced enzyme was indistinguishable from the wild-type enzyme. A homogeneous phospholiphase A preparation was obtained in high yield from overproducing bacteria, using the zwitterionic detergent C12-Sulfobetaine and anion-exchange chromatography. Detergent gradients were found to exert great influence on the elution characteristics. Considerations for the choice of optimal detergent gradients are discussed. The purified enzyme migrated as a single 29-kDa band in SDS/polyacrylamide gels, and required Ca(II) for activity. Maximum activity was displayed by enzyme samples taken from solutions with detergent concentrations near the critical micelle concentration. However, upon switching from high to optimal detergent concentration, maximum activity was restored in several hours, probably reflecting a slow conformational transition of the protein. Because the final pure protein was found to hydrolyze phospholipids in the intact erythrocyte membrane, a densely packed bilayer, we assume that this protein is in its biological native state.     

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5''-nucleotidase.

An alkaline 5'-nucleotidase with properties similar to those of membrane-bound 5'-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.