Isolation and identification of yolk proteins in Indian major carp,Labeo rohita

Two yolk proteins (YP1 and YP2) from the ovaries of Indian major carp, Labeo rohita were isolated by gel filtration and partially characterized by the use of hydroxyapatite ultrogel column in conjunction with native PAGE. On native PAGE YP1 gave a single protein band, whereas YP2 of gel filtration revealed the contamination of YP1, which was removed by adsorption chromatography on hydroxyapatite ultrogel and then the YP2 was the purified one as judged by electrophoresis. Both YP1 and YP2 also stained for lipid and contained alkalilabile phosphorus. Therefore, both yolk proteins were lipophosphoprotein. The molecular weights of YP1 and YP2 were 620 kDa and 225 kDa respectively as determined by gel filtration on Sepharose 4B. When YP1 and YP2 were compared in relation to some physicochemical characteristics with yolk proteins of other oviparous vertebrates including fish, they were lipovitellin like. Antiserum to YP2 crossreacted with YP2 and vitellogenin suggesting that YP2 was the cleaved product of vitellogenin. Anti-YP2 antiserum was not crossreacted with native YP1, whereas reduced and/or denatured YP1 was crossreacted indicating the presence of antigenic determinants in the inner core region of YP1 polypeptide.     

High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone III

ABSTRACT. Tritiated 10,11-epoxyfarnesyl diazoacetate (EFDA), a photoaffinity label, can be covalently attached to the binding site of a JH-III-specific binding protein in the haemolymph of Locusta migratoria migratorioides (R & F). The specificity of the binding of EFDA to the binding protein is verified by displacement with excess unlabelled JH-III, and EFDA can be used to identify the binding protein in native pore-limiting gradient poly(acrylamide) gel electrophoresis (PAGE) and sodium dodecyl sulphate-PAGE. The native binding protein has a molecular weight of 575,000 and is composed of seemingly identical subunits of molecular weight 81,000.
Three other high-molecular weight serum proteins are identified by native PAGE: a lipophorin, composed of two kinds of apolipophorins, a larval storage protein and a cyanoprotein. The molecular weights and subunit structures of these proteins are investigated, but none of these other high-molecular weight proteins bind JH-III to an appreciable extent.     

Identification of a Major Xylanase from <Emphasis Type="Italic">Aspergillus flavus</Emphasis> as a 14-kD Protein

Aspergillus flavus K49 secreted at least two xylanase activities when grown on a medium containing larch (wood) xylan as a sole carbon source. Enzyme activity was assayed using an agar medium containing Remazol Brilliant Blue R conjugated oat spelt xylan as substrate. Crude enzyme preparations were inhibited by Hg⁺², with an ED₅₀ of 17.5 mM and maximum inhibition of 83% at 50 mM. A concentrated sample of A. flavus K49 xylanase preparation was subjected to gel filtration chromatography on a P-30 column. A small protein peak coinciding with the major peak of xylanase activity was separated from the other secreted fungal proteins. An additional peak of xylanase activity was observed in fractions containing multiple fungal proteins. Analysis by denaturing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of fractions containing the smaller molecular weight xylanase revealed a major and minor protein band in the vicinity of 14 kD. Analysis of these same fractions by acidic native PAGE revealed a single band. Confirmation of identity for the isolated xylanase was provided by isolation of a protein band from a SDS–PAGE gel, followed by trypsin digestion/analysis by tandem mass spectrometry. Comparison of the peptide library derived from this protein band with sequence data from the A. oryzae genomic data base provided a solid match with an endo-1,4-β-xylanase, XlnA. This identification is consistent with a low molecular weight protein associated with the major xylanolytic activity. XlnA may be a highly mobile (diffusible), plant wall hemicellulose degrading factor with significant activity during plant infection.     

鹅IL-2基因在大肠杆菌中的表达及其可溶性单体的分离

将去除信号肽编码序列的鹅IL-2基因克隆到原核表达载体pET-28a (+), 构建了重组表达质粒pET-28a (+)-goIL-2, 转化大肠杆菌BL21(DE3)感受态细胞, 经IPTG诱导, 实现了重组鹅IL-2(rgoIL-2)蛋白在大肠杆菌中的表达。SDS-PAGE和Western-blotting分析显示, 表达蛋白的分子量约为15.0 kD, 能被抗鹅IL-2单克隆抗体特异识别。可溶性分析表明表达蛋白大部分以包涵体形式存在, 部分以可溶形式存在, 非变性电泳可见可溶性蛋白存在单体和多聚体组分。镍柱亲和层析法纯化的rgoIL-2蛋白过滤后, 利用?KTA FPLC(快速蛋白分离纯化系统)进行逐级分离, 非变性电泳可见单一的鹅IL-2可溶性蛋白单体。体外生物学活性分析显示鹅IL-2可溶性蛋白单体能刺激鹅淋巴细胞增殖。这为进一步研究鹅IL-2的生物学功能及其临床应用奠定基础。     

鹅IL-2基因在大肠杆菌中的表达及其可溶性单体的分离

将去除信号肽编码序列的鹅IL-2基因克隆到原核表达载体pET-28a (+), 构建了重组表达质粒pET-28a (+)-goIL-2, 转化大肠杆菌BL21(DE3)感受态细胞, 经IPTG诱导, 实现了重组鹅IL-2(rgoIL-2)蛋白在大肠杆菌中的表达。SDS-PAGE和Western-blotting分析显示, 表达蛋白的分子量约为15.0 kD, 能被抗鹅IL-2单克隆抗体特异识别。可溶性分析表明表达蛋白大部分以包涵体形式存在, 部分以可溶形式存在, 非变性电泳可见可溶性蛋白存在单体和多聚体组分。镍柱亲和层析法纯化的rgoIL-2蛋白过滤后, 利用?KTA FPLC(快速蛋白分离纯化系统)进行逐级分离, 非变性电泳可见单一的鹅IL-2可溶性蛋白单体。体外生物学活性分析显示鹅IL-2可溶性蛋白单体能刺激鹅淋巴细胞增殖。这为进一步研究鹅IL-2的生物学功能及其临床应用奠定基础。     

Isolation of human alpha-fetoprotein in two fractionation steps and demonstration of homogeneity.

Human alpha-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pI') of 4.85, molecular radius of 3.0 nm and a valence (net H+/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.     

Isolation of Human α-Fetoprote in Two Fractionation Steps and Demonstration of Homogeneity

Human α-fetoprotein (hAFP) has been isolated from cord serum in 40% yield using an isolation procedure consisting of only two major steps: affinity chromatography followed by preparative polyacrylamide gel electrophoresis (PAGE). The final product appeared homogeneous on the basis of five independent criteria for purity. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) demonstrated a single polypeptide chain with molecular weight of 71,000. The protein exhibited an apparent isoelectric point (pÍ) of 4.85, molecular radius of 3.0 nm and a valence (net H⁺/molecule) of 21.9 derived from computation of analytical PAGE data. The two-step isolation procedure made it possible for a single operator to isolate milligram amounts of hAFP in a matter of weeks.     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

The effect of incorporating carbon nanotubes (CNTs) in the gel matrix on the electrophoretic mobility of proteins based on their molecular weight differences was investigated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). More specifically, a reduction in standard deviation in the molecular weight calibration plots by 55% in the case of multiwalled carbon nanotubes (MWCNTs) and by 34% in the case of single-walled carbon nanotubes (SWCNTs) compared with that of pristine polyacrylamide gels was achieved after incorporating an insignificant amount of functionalized CNTs into the gel matrix. A mechanism based on a more uniform pore size distribution in CNT modified polyacrylamide gel matrix is proposed. Furthermore, the impact of SWCNTs and MWCNTs on the mobility of proteins in different molecular weight regimes at a given acrylamide concentration offers a tunable gel matrix in terms of the selection of molecular weight ranges of proteins. The robustness and excellent reproducibility of the CNT–PAGE protocol are expected to have a significant impact on the molecular weight determination of newly isolated proteins.     

Purification of monomeric prolactin charge isoform from buffalo pituitaries

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S-200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti-buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.     

Identification and characterization of latex-specific proteins in opium poppy

Latex from the opium poppy, Papaver somniferum L., was analyzed by polyacrylamide gel electrophoresis (PAGE). Two latex-specific bands were identified in protein samples of poppy latex using one-dimensional native PAGE. Second dimension analysis with SDS-PAGE indicates that these proteins have a relative molecular weight of approximately 20 kilodaltons. We have termed these polypeptides the major latex proteins (MLPs). Polyclonal antibodies prepared against the MLPs were used to probe protein gel blots of latex and poppy tissues known to lack laticifers. Laticifer-free tissues showed no reaction with anti-MLP immunoglobulin G indicating that MLPs are found only in poppy latex. MLP distribution was also examined in mature opium poppy tissues by immunocytochemistry. Laticifers were differentially labeled by fluorescein isothiocyanate secondary labeling of anti-MLP immunoglobulin G and could easily be identified in both transverse and longitudinal section. Fractionation studies of isolated latex showed that MLPs are concentrated in the latex cytosol and not in alkaloidal vesicles. Analysis of latex proteins by conventional two-dimensional electrophoresis indicates that the two MLP bands are composed of several distinct polypeptides with similar relative molecular weights. The pIs of these molecules range from 6.0 to 3.5. The role(s) of MLPs in laticifer metabolism has not been determined.     

Analysis of Streptomyces coelicolor membrane proteome using two-dimensional native/native and native/sodium dodecyl sulfate gel electrophoresis

Analysis of the oligomeric state of a protein may provide insights into its physiological functions. Because membrane proteins are considered to be the workhorses of energy generation and polypeptide and nutrient transportation, in this study we characterized the membrane-associated proteome of Streptomyces coelicolor by two-dimensional (2D) blue native/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), high-resolution clear native/native PAGE, and native/SDS–PAGE. A total of 77 proteins were identified, and 20 proteins belonging to 15 complexes were characterized. Moreover, the resolution of high-resolution clear native/SDS–PAGE is much higher than that of blue native/SDS–PAGE. OBP (SCO5477) and BldKB (SCO5113) were identified as the main protein spots from the membrane fractions of S. coelicolor M145, suggesting that these two proteins are involved in extracellular peptide transportation. These two transporters exhibited multiple oligomeric states in the native PAGE system, which may suggest their multiple physiological functions in the development of S. coelicolor.     

多种小麦种子储藏蛋白的电泳分析

采用不同的提取液,对１０个小麦品种的非酶功能性种子储藏蛋白进行提取,分别进行梯度凝胶电泳分析。电泳依据提取液的不同,分别采用酸性或碱性系统。对酸性凝胶催化系统,采用Ａｐ－Ｖｃ－ＦｅＳＯ４系统代替Ｈ２Ｏ２－Ｖｃ－ＦｅＳＯ４系统,克服了酸性凝胶的不足,提高了凝胶的性质性能并使之容易操作。应用新的催化系统配制的酸性梯度胶,提高了分辨率。并初步尝试以酸性系统分析种子谷蛋白,获得了成功,经过对不同提取液蛋白     

Purification of Monomeric Prolactin Charge Isoform from Buffalo Pituitaries

Abstract

A standard protocol for isolation of buffalo prolactin (buPRL) was modified at the alcohol precipitation step. This modification could separate lower molecular weight prolactin from the higher molecular weight prolactin (PRL). Reloading the prolactin onto a Sephacryl S‐200 gel purified the buPRL monomer. The purity of buPRL monomer was confirmed by 15% SDS PAGE. The buPRL monomer was >90% pure. It was characterized by specific anti‐buPRL serum in ELISA and Western blot. A native PAGE of the PRL showed three charge isoforms. A protocol was standardized to separate prolactin monomeric least acidic isoforms using an anion exchanger.     

Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis.

Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N terminus in an Edman degradation analysis. MDH activity was detected in purified preparations by activity staining of gels in the direction of malate oxidation. PAGE and gel filtration (Sephadex G-100) analyses showed the native enzyme to be a dimer composed of identical subunits both at room temperature and at 4 degrees C. The molecular weight of the native enzyme as estimated by gel filtration was 77,000 and by gradient PAGE was 74,000. The subunit molecular weight as estimated by SDS-gradient PAGE was 37,500. N-terminal sequences of MDHs from C. vibrioforme, Chlorobium tepidum, and Heliobacterium gestii are presented. There are obvious key sequence similarities in MDHs from the phototrophic green bacteria. The sequences presented probably possess a stretch of amino acids involved in dinucleotide binding which is similar to that of Chloroflexus aurantiacus MDH and other classes of dehydrogenase enzymes but unique among MDHs.     

Partial Purification of Proteinase K Inhibitors from Liquid-Cultured Mycelia of the White Rot Basidiomycete Trametes versicolor

Novel protease inhibitors were isolated from liquid-cultured mycelia of the white rot fungus Trametes versicolor. Two bands of antiproteinase K activity, TvPI-A and TvPI-B, were detected in the crude cell extract by native polyacrylamide gel electrophoresis (PAGE). Proteins corresponding to TvPI-A were purified by heat treatment, anion-exchange chromatography, and gel filtration. Sodium dodecyl sulfate (SDS)-PAGE demonstrated the presence of three proteins with molecular masses of 14.5, 16.6, and 20 kDa, respectively. T. versicolor protease inhibitors suppressed the activity of proteinase K and, to a smaller extent, of Carlsberg subtilisin, whereas trypsin and chymotrypsins were not inhibited. The inhibitors were acidic proteins and showed remarkable heat stability. To our knowledge, this is the first report about proteinase K inhibitors from fungi.     

Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract.

A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native/SDS-2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor alpha chain (IL-2Ralpha) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2/IL-2-2Ralpha complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions.     

Phaseolus coccineus L. Storage Proteins. Extraction and Characterization

Phaseolus coccineus storage globulins were extracted from mature cotyledons, purified and characterized. Three major proteins were separated. A component showing erythroagglutinating activity was thoroughly purified by thyroglobulin-Sepharose chromatography. The relative molecular masses of the three fractions are Mr = 330, 178, and 500 kDa as determined by polyacrylamide gel electrophoresis (PAGE). They correspond to the proteins found in other systems and classified as phytohaemagglutinin (PHA), vicilin and legumin, respectively. Electrophoretic analyses under denaturating conditions (SDS-PAGE) evidenced the major subunits for the three proteins. Isoelectrofocusing of the isolated proteins indicated a large heterogeneity for vicilin. Part I.     

Human lymphocyte membrane proteins treated with neuraminidase

Human peripheral blood lymphocytes were surface-iodinated, treated with neuraminidase from Vibrio cholerae and lysed with non-ionic detergent. In addition, surface membrane fractions were isolated from surface-iodinated cells in the absence of detergents and treated with neuraminidase after membrane isolation. The effect of neuraminidase treatment on the membrane proteins was studied by two-dimensional gel electrophoresis. One surface-labelled protein of 45 000 molecular weight which is characterized by its association with the detergent-resistant matrix of the cells and by its specific enrichment in an isolated membrane fraction, was found to be particularly sensitive to neuraminidase treatment both of intact cells and isolated membranes. A prominent labelled protein of apparent molecular weight of 60 000 is observed in the soluble fraction after neuraminidase treatment of intact cells. The analogous protein is detected when isolated membrane fractions are treated with neuraminidase.     

金鱼(红龙睛)次黄嘌呤-鸟嘌呤磷酸核糖转移酶的分离及特性初步研究

 从金鱼肝脏分离并部分纯化了HGPRT。初步结果表明,该酶的分子量为78,000左右。由3个亚基组成,每个亚基的分子量约为27,000。金鱼的HGPRT很稳定,在85℃加热10分钟,至少保留80％的酶活性。该酶与PRPP的反应产物在淀粉凝胶电泳图谱上出现两条反应带。     

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.