Gel filtration high performance liquid chromatography of envelope polypeptide variants of herpes simplex type 1 strains

High performance liquid chromatography (HPLC) with a TSK-4000SW gel filtration column was used to compare envelope polypeptides from four strains of herpes simplex virus type 1 (HSV-1). The chromatographic profiles demonstrated polypeptide variability among three clinical strains and the wild-type F strain. Radioimmunoprecipitation of the HPLC fractions with polyclonal anti-HSV-1 followed by SDS-polyacrylamide gel electrophoresis (PAGE) of the immunoprecipitates revealed molecular weight differences of various polypeptides in fractions from the area containing major peaks. This HPLC method could prove useful for the analysis of polypeptide polymorphism in clinical isolates of HSV-1, as well as in other viruses.     

Specific Proteins in the Indirect Somatic Embryogenesis of Freesia Refracta

IntroductionHaberlandt[1]proposed the conception of plantso-matic embryo in 1902, which states that plant cellsexhibit totipotency; each cell could divide ceaselesslyand eventually develops into a mature plantlet. Allthese provide a theoretical evidence for somatic embryo-genesis. The related researches reveal thatplantembry-ogenesis occurs as a result of the selective expression ofsets of genes that allowthe synthesis of special proteinsin a temporal and spatial sequential manner under themut…     

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.     

Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure

We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria. Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications. The trains were found to be regenerated when rerunning individual polypeptide spots. The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al. (Protein Eng. 1990, 4, 155-161). The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse.     

Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS

Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.     

The Properties of an HBV Surface Antigen Protein Carrying the Binding Site for the Receptor of Hepatocytes——Its Formation of Surface Antigen Particles and Secretion From Discrete Cell Lines

A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination. When expressed in monkey kidney cell line COS-M6, this gene product (S309 protein) formed surface antigen (HBsAg) particles and secreted from the cells. It was stable within the cells and in the culture medium and could be immunoprecipitated with antisera directed against plasma-derived HBsAg or synthetic preS1 polypeptide. Isopycnic CsCl gradient centrifugation showed that the density of S309 protein particles (1.25 g/ml) was slightly higher than that of S protein particles. The S309 protein was readily secretable from hepatoma cell lines, and the amount secreted was comparable to that of the S protein. By contrast, only about 10% of the S309 protein was secreted from COS-M6 cells, and its appearance in culture medium was delayed. The efficiency of the secretion of the S309 protein can b     

Noncovalent interactions in human plasma proteins analyzed by the comparison of nondenaturing and denaturing micro-2-D gel electrophoresis patterns after polypeptide assignment using matrix-assisted laser desorption/ionization-mass spectrometry and peptide mass fingerprinting

Previously, we reported the analysis of human plasma proteins by 2-DE under nondenaturing conditions (Type-I 2-DE) followed by the assignment of stained spots using MALDI-MS and PMF [1]. Here, we employ 2-DE conditions modified only in the second-dimensional separation; SDS was added in the gradient slab gel aiming to dissociate noncovalently bound proteins/polypeptides (Type-II 2-DE). Totally 169 CBB-stained spots on a micro-2-DE gel were numbered and subjected to polypeptide assignment using MALDI-MS-PMF. One hundred sixty spots out of the 169 provided significant match (p <0.05) with polypeptides in databases. Comparisons of the results of polypeptide assignment on the two 2-DE patterns indicated that 10 polypeptides in 20 stained spots on the Type-I 2-DE pattern [1] shifted toward low-molecular-weight positions on the Type-II 2-DE pattern, demonstrating the presence of noncovalent interactions. Seventeen polypeptides in 38 stained spots were only assigned on the Type-II 2-DE gel, which could mostly be accounted for by the disruption of noncovalent protein-protein interactions in the presence of SDS, i.e., protein/polypeptide complexes which might form smear bands on the Type-I 2-DE gel dissociate to form clear spots on the Type-II 2-DE gel. The method employed here, comparisons of nondenaturing and denaturing 2-DE maps with polypeptide assignment by MALDI-MS-PMF, would enable the simultaneous detection of multiple noncovalent interactions in complex protein/polypeptide systems.     

Experimental Studies with a Bonded N-acetylaminopropylsilica Stationary Phase for the Aqueous High Performance Exclusion Chromatogrpahy of Polypeptides and Proteins

Abstract

The potential of the micoparticulate, chemically bonded N-acetylaminopropylsilica stationary phase of nominal pore diameter of 100 angstroms in the high speed gel permeation chromatography of polypeptides and small proteins has been further investigated. The influence of ionic strength on the elution behaviour of a selected group of polypeptides and proteins on this bonded hydrophilic support has been examined. The results obtained with this porous, microparticulate bonded ‘amide’ phase silica support, packed into standard analytical-size stainless steel HPLC columns, indicate that milligram quantities of polypeptides and protein swith molecular mass up to 45,000-50,000 daltons can be efficiently fractionated with excellent recoveries of biological activities. The role of silica-based sorbents in the gel permeation fractionation of polypeptide and protein hormones, including those of pituitary and hypothalamic origin, is discussed.     

Polypeptide subunits of dynein 1 from sea urchin sperm flagella.

A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000--350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4 degrees C with a solution containing 0.6 M NaCl, ph 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21S and a minor peak at 12--14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000--122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000--24,000) cosediment with the 21S peak. The heavy chain composition of the 12--14S peak is more complex, all eight heavy chains occurring approximately the same ratios as occur in intact axonemes.     

Separable Forms of Radioiodinated Ovine Lutropin (oLH) Subunits,Fractionated by Anion Exchange High Performance Liquid Chromatography and Analyzed by Radioimmunoassay

Abstract

Radioiodinated oLH α and oLH ß subunits were fractionated with the aid of high performance liquid chromatography (HPLC) using a Waters Protein Pak DEAE 5PW anion exchange column. The content of these subfractions differed in their binding maxima to their respective subunit antisera. An increase of the pH from 6.5 to 7.5 and 8.5 affected the chromatographic profile of 8-week-old radioiodinated α-subunit. Overall, material from the various radioactive peaks exhibited binding to ß-subunit antiserum in the range of 32.0% - 81.0%, depending on the storage time of the tracer and the pH. Shifting strategies, we either applied the labelled subunits to a Pharmacia gel filtration column or subjected them to cellulose adsorption prior to HPLC. The radioiodinated α- and β-subunits subjected to HPLC after gel filtration were both eluted in only one peak with respective immunoreactivities of 46.6% and 73.2%.

When radioiodinated β-subunit was applied first to a cellulose column and then to HPLC, the chromatographic profile showed two radioactive peaks with retention times of 5 min (73.2% immunoreactivity) and 7.5 min (43.0% immunoreactivity), respectively.

It was concluded that an 8-week-old-tracer i s useful in such studies, owing to its highly stable immunoreactivity after repurification on an anion exchange HPLC.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Two-dimensional electrophoresis of the total polypeptides in ripe red grape berries.

The total polypeptide composition of mature grape berries was analyzed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis (isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension), followed by Coomassie Blue and nitrate silver staining, respectively. Adapted methods for total protein preparation of grapes and for two-dimensional gel electrophoretic separation of polypeptides are presented. The grape patterns presented up to 52 fractions with Mrs ranging from 15,000 to 110,000. The polypeptides displayed pIs from 4.6 to 7.3. A group of spots from Mr 28,000 to 83,000 and with a pI from 4.6 to 5.4 was strongly silver stained. The Mr 28,000 spot, pI 4.6, was revealed to be a complex of four fractions. Reproducible separations were obtained with the different carrier ampholyte mixtures tested.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

Polypeptide changes induced by salt stress, water deficit, and osmotic stress in barley roots: a comparison using two-dimensional gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments (24 h) of salt stress, water deficit (desiccation), and osmotic stress (polyethylene glycol and mannitol) on protein synthesis in roots of barley seedlings (Hordeum vulgare L. cv. CM 72). These comparisons were made to determine if the polypeptides of Mr 26,000 and 27,000 and pI of 6.3 and 6.5 that were observed previously to increase significantly with salt stress (Plant Physiol. 1987, 83 517-524) also increased with water deficit and osmotic stress. The polypeptide patterns for control- and stress-treated plants were qualitatively similar, but the net synthesis of a number of polypeptides was quantitatively altered by each of the stress treatments. Of the polypeptide changes induced by the stress treatments, many were unique to a specific stress. Other polypeptide changes were common between two or more of the stress treatments. Only one polypeptide change, a decrease, was common to all of the stress treatments. An important finding was that polypeptides that increased significantly in response to salt stress did not increase in response to water deficit or osmotic stress.     

Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system

Summary A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.     

PHOTORECEPTOR PROTEINS AND PIGMENTS IN THE PARAFLAGELLAR BODY OF THE FLAGELLATE Euglena gracilis

Abstract— –The presumed photoreceptor for phototaxis, the paraflagellar body, in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization, fast protein liquid chromatography (FPLC) analysis yielded four major protein fractions with the chromophoric groups still attached. Fluorescence spectra showed that three fractions had excitation peaks at 380 nm and emission peaks around 450 nm indicative of pterins, while the fourth chromoprotein had a fluorescence emission at 520 nm and an excitation peak at 450 nm, indicative of a flavin. The separated proteins were analyzed by gel electrophoresis: the pterin binding proteins have apparent molecular masses between 27 000 and 31 600 and the flavin binding protein has an apparent molecular mass of 33 500.     

Analysis of E. coli soluble proteins by non‐denaturing micro 2‐DE/3‐DE and MALDI‐MS‐PMF

Escherichia coli (strain K‐12)‐soluble proteins were analyzed by nondenaturing micro 2‐DE and MALDI‐MS‐PMF. The reported conditions of nondenaturing IEF in agarose column gels [Jin, Y., Manabe, T., Electrophoresis 2009, 30, 939–948] were modified to optimize the resolution of cellular soluble proteins. About 300 CBB‐stained spots, the apparent molecular masses of which ranged from ca. 6000 to 10 kDa, were detected. All the spots on two reference 2‐DE gels (one for wide mass range and one for low‐molecular‐mass range) were numbered and subjected to MALDI‐MS‐PMF for the assignment of constituting polypeptides. Most of the spots (310 spots out of 329) provided significant match (p<0.05) with polypeptides in Swiss‐Prot database and totally 228 polypeptide species were assigned. Activity staining of enzymes such as alkaline phosphatase and catalases was performed on the 2‐DE gels and the locations of the activity spots matched well with those of the MS‐assigned polypeptides of the enzymes. Most of the polypeptides with subunit information in Swiss‐Prot (119 polypeptides as homo‐multimers and 25 as hetero‐multimers out of the 228), such as pyruvate dehydrogenase complex which is composed of three enzymatic components, were detected at the apparent mass positions of their polymers, suggesting that the proteins were separated retaining their subunit structures. When a nondenaturing 2‐DE gel was vertically cut into 2 mm strips and one of the strips was subjected to a third‐dimension micro SDS‐PAGE (micro 3‐DE), about 190 CBB‐stained spots were detected. The assignment of the polypeptides separated on the 3‐DE gel would further provide information on protein/polypeptide interactions.     

Dependence on effective saturation of numbers of singlet peaks in one- and two-dimensional separations

The average numbers of singlet peaks in one-dimensional (1D) and two-dimensional (2D) separations of randomly distributed peaks are predicted by statistical-overlap theory and compared against the effective saturation. The effective saturation is a recently introduced metric of peak crowding that is more practitioner-friendly than the usual metric, the saturation. The effective saturation absorbs the average minimum resolution of statistical-overlap theory, facilitating the comparison of 1D and 2D separations by traditional metrics of resolution and peak capacity. In this paper, singlet peaks are identified with maxima produced by a single mixture constituent. Their effective saturations are calculated from published equations for the average minimum resolution of 1D singlet peaks, and from equations derived here for the average minimum resolution of 2D singlet peaks. The fractions of peaks that are singlets in 1D and 2D separations are predicted by statistical-overlap theory as functions of saturation but are compared as functions of effective saturation. The two fractions differ by no more than 0.033 at any effective saturation between 0 and 6, when the distribution of peak heights is exponential and the edge effect is neglected. This result shows that 1D and 2D separations of randomly distributed peaks are about the same in their ability to separate singlet peaks as maxima, when assessed relative to effective saturation. Empirical equations in effective saturation are reported for the fractions of peaks that are singlets. It is argued that the effective saturation is a good metric for comparing separations having different average minimum resolutions.     

梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.