Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.     

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.     

Modification of simian virus 40 protein A.

The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.     

Indirect tissue electrophoresis: a new method for analyzing solid tissue protein

1. The eye lens core (nucleus) has been a valuable source of molecular biologic information. 2. In these studies, lens nuclei are usually homogenized so that any protein information related to anatomical subdivisions, or layers, of the nucleus is lost. 3. The present report is of a new method, indirect tissue electrophoresis (ITE), which, when applied to fish lens nuclei, permitted (a) automatic correlation of protein information with anatomic layer, (b) production of large, clear electrophoretic patterns even from small tissue samples and (c) detection of more proteins than in liquid extracts of homogenized tissues. 4. ITE seems potentially applicable to a variety of solid tissues.     

Assay of neomycin phosphotransferase activity in cell extracts.

A simple method for the measurement of neomycin phosphotransferase (NPT) activity in crude extracts of eukaryotic cells is described. This method is based on the elimination of interfering phosphorylated proteins by using phenol-chloroform extraction. This solution phase assay allows the detection of greater than or equal to 0.01 ng of NPT in the crude cell extract. Rapid screening of a large number of cell cultures generated in gene-transfer experiments, using NPT as a selective marker, is made possible by this simple technique. Further, the promoter strength of vector constructs used in gene therapy may also be estimated by this procedure.     

Protein carboxyl methyltransferase from cow eye lens

Protein carboxyl methyltransferase activity (S-adenosyl-L-methionine: protein carboxyl-0-methyltransferase; E.C. 2.1.1.24) has been detected in crude soluble extracts of cow eye lens. The activity incorporates methyl groups from S-adenosyl-L-methionine into endogenous lens proteins in vitro, and several of these species co-migrate electrophoretically with lens crystallins. A 2600-fold purification of the enzyme free of endogenous substrates was achieved by gel filtration and affinity chromatography. The lens methyltransferase has a native molecular weight of approximately 27,000, and catalyzes the substoichiometric incorporation of highly alkali-labile methyl ester groups into a broad range of protein substrates. The lens enzyme appears to be similar to that found in human erythrocytes, which specifically recognizes and modifies D-aspartic acid residues in aged proteins in a postulated degradative or racemization-repair pathway (McFadden, P.N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464).     

线虫surface coat proteins提取方法的建立与双向电泳分析

目的:建立一套适用于蛋白质双向电泳体系的线虫surface coat proteins(SCPs)样品制备技术,为今后研究线虫surfacecoat蛋白质组学及线虫病理生理学奠定基础.方法:以秀丽隐杆线虫(Caenorhabditis elegans)为研究材料,对比和分析不同的蛋白提取沉淀方法,进而采用SDS-PAGE电泳技术和双向电泳技术对所提蛋白进行评价.结果:通过35%乙醇结合TCA-丙酮沉淀法获得的质量较好的线虫SCPs,在12%的SDS-PAGE分析中该法提取的蛋白背景浅,蛋白条带多且清晰尖锐,含有丰富的蛋白信息量.通过双向电泳分析,可从提取的蛋白中鉴定出清晰蛋白点400多个.随机选择5个蛋白斑点,进行基质辅助激光解吸电离飞行时间质谱鉴定,鉴定得到高度匹配的已知线虫蛋白质2个.结论:所建立的方法可为今后研究线虫surface coat蛋白质组学及线虫病理生理学提供重要工具.     

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI-TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.     

Crystallins of the octopus lens. Recruitment from detoxification enzymes.

The eye lens crystallins of the octopus Octopus dofleini were identified by sequencing abundant proteins and cDNAs. As in squid, the octopus crystallins have subunit molecular masses of 25-30 kDa, are related to mammalian glutathione S-transferases (GST), and are encoded in at least six genes. The coding regions and deduced amino acid sequences of four octopus lens cDNAs are 75-80% identical, while their non-coding regions are entirely different. Deduced amino acid sequences show 52-57% similarity with squid GST-like crystallins, but only 20-25% similarity with mammalian GST. These data suggest that the octopus and squid lens GST-like crystallin gene families expanded after divergence of these species. Northern blot hybridization indicated that the four octopus GST-like crystallin genes examined are lens-specific. Lens extracts showed about 40 times less GST activity using 1-chloro-2,4-dinitrobenzene as substrate than liver extracts of the octopus, indicating that the major GST-like crystallins are specialized for a lens structural role. A prominent 59-kDa crystallin polypeptide, previously observed in octopus but not squid and called omega-crystallin (Chiou, S.-H. (1988) FEBS Lett. 241, 261-264), has been identified as an aldehyde dehydrogenase. Since cytoplasmic aldehyde dehydrogenase is a major protein in elephant shrew lenses (eta-crystallin; Wistow, G., and Kim, H. (1991) J. Mol. Evol. 32, 262-269) the octopus aldehyde dehydrogenase crystallin provides the first example of a similar enzyme-crystallin in vertebrates and invertebrates. The use of detoxification stress proteins (GST and aldehyde dehydrogenase) as cephalopod crystallins indicates a common strategy for recruitment of enzyme-crystallins during the convergent evolution of vertebrate and invertebrate lenses. For historical reasons we propose that the octopus GST-like crystallins, like those of the squid, are called S-crystallins.     

Investigation of the mechanisms of crystallin aggregation induced by pulsed laser UV irradiation at 308 nm

The results of the investigations of photoaggregation of the main eye lens proteins alpha-, beta- and gamma-crystallins and the model protein carbonic anhydrase in response to pulsed irradiation by a XeCI laser at 308 nm in the wide range of pulse energy densities (w) and pulse repetition rates (F) have been reviewed. A nonlinear dependence of aggregation efficiency on the values of w, F, and the concentration of protein solution was found. A theoretical model that qualitatively describes the experimental results was developed. The aggregation of N-amino-arm truncated beta A3-crystallin was analyzed. It was found that the loss of the N-amino-arm as a result of mutation or eye lens aging increases the probability of UV-induced beta-crystallin aggregation, thereby increasing the predisposition of eye lens to senile cataract. The influence of some short-chain peptides on the aggregation efficiency of beta-crystallin and beta-crystallin in solution with alpha-crystallin was investigated. Based on the results obtained, a combination of peptides (called "a new preparation") was found that most effectively delays the crystallin aggregation. The preparation has been probed on experimental animals. The trials showed that the preparation increases the delay in the development of UV-induced cataract in rats. The possibility of designing a drug for the prophylaxis of the development of cataract in humans based on this preparation is discussed.     

The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.     

Differential extraction of sediment phosphates with NTA solutions

An extraction technique is described for the separation of iron- and calcium-bound phosphate. The iron-bound phosphate is extracted with a 0.05 M solution of Ca-NTA under reducing conditions. Iron, also, is brought into solution, which is an advantage over the NaOH extraction. The calcium-bound phosphate is extracted with a 0.05 M solution of Na-NTA. The NTA also extracts humic compounds. Organic phosphate compounds can be measured in the NTA extracts, unlike the NaOH or H₂SO₄ extracts such as are used in the (modified) Jackson procedure.Examples of some test compound extractions and of a calcareous sediment are given.     

A contribution to the standardization of methods for the preparation of seed proteins ofAllium cepa L.

The study of certain conditions for the extraction of seed proteins ofAllium cepa revealed that the best extractibility of proteins is obtained by the use of a buffered physiological solution at 20 °C in comparison with TRIS-glycine buffer at 5 °C. Using potassium phosphate buffer with 0.01 M mercaptoethanol and 0.4 M NaCl, an amount of proteins by up to 25 per cent higher passes into solution as compared with the physiological solution, but these extracts are unsuitable for the electrophoretic separation in polyacrylamide gels. The defatting of the seed meal under low temperature did not affect the qualitative composition of the protein complex studied, the addition of a 1 per cent soluble starch to the polyacrylamide gel. improved its resolution.     

不同提取液提取水稻幼苗质外体蛋白效果的比较

提取植物组织质外体蛋白质的主要困难是提取效率低且易被细胞质蛋白污染。为解决上述问题,以12天93-11水稻幼苗为试验材料,使用3种含不同浓度钾和钙离子的缓冲液作为提取液进行提取效果比较。3种提取液的相同成分都是0.1mol/L Tris-HCl pH 7.6,1mmol/L PMSF,区别点在于:Buffer A含0.2mol/L KCl;Buffer B含0.2mol/L CaCl₂;Buffer C含0.1mol/L KCl和0.1mol/L CaCl₂。结果表明,Buffer A的蛋白产率达到了(0.49±0.07)mg/g FW(叶片)和(0.83±0.06)mg/g FW(根部),比Buffer B和Buffer C分别提高了122.7%和53.1%(叶片)以及102.4%和59.6% (根部)。六磷酸葡萄糖脱氢酶活性检测的结果表明在这些蛋白质提取物中细胞质蛋白的污染率很低,可以控制在1%以下。这些实验结果说明通过优化提取液,建立了有效提取水稻幼苗质外体蛋白质的方法,可应用于植物质外体蛋白质组学研究。     

Chloroform-Assisted Phenol Extraction Improving Proteome Profiling of Maize Embryos through Selective Depletion of High-Abundance Storage Proteins

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.     

Simple and rapid method for disruption of bacteria for protein studies.

A simple and rapid method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with acetone followed by sodium dodecyl sulfate extraction of cellular proteins. Polyacrylamide gel electrophoresis revealed that the protein composition of extracts made by this method was comparable to that of extracts made by established methods, namely, sonication and agitation with beads. This technique has been successfully applied to the extraction of proteins from a wide variety of bacteria, including pathogens.     

Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts

Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli; phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of E. coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H](-) ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.     

Simple and rapid method for disruption of bacteria for protein studies

A simple and rapid method was developed for the extraction of proteins from both pathogenic and nonpathogenic bacteria. The method involves the treatment of cells with acetone followed by sodium dodecyl sulfate extraction of cellular proteins. Polyacrylamide gel electrophoresis revealed that the protein composition of extracts made by this method was comparable to that of extracts made by established methods, namely, sonication and agitation with beads. This technique has been successfully applied to the extraction of proteins from a wide variety of bacteria, including pathogens.     

Rapid method for the determination of lysine in cereal grains without hydrolysis.

A rapid spectrophotometric method for lysine determination in cereal grains is reported. The reagent is sodium dinitrobenzene sulfonate (DNBS). The absorbance of the acid solution of the DNBS derivative is read at 385 nm. Under prescribed conditions, the reagent is sensitive and specific for free or protein-bound lysine. The conditions: For rice, the reaction medium is a urea-phosphate-HgCl₂ solution (pH 10.5); reaction at 60°C for 1 hr. For other grains, the reaction medium is a urea-phosphate-phenylmercuric chloride (PMC) solution (pH 12.3); reaction at 40°C for 1 hr. Interferences are eliminated by ether extraction after color development and acidification and addition of formic acid and the sulfhydryl masking agents, HgCl₂ or phenylmercuric chloride (PMC). NaOH extracts of cereal proteins are used for analysis. Values are in agreement with those of the ion-exchange amino acid analyzer.     

Isolation of enamelinlike proteins from blue shark (Prionace glauca) enameloid

A sequential dissociative extraction scheme was used to extract proteins from developing Blue Shark enameloid. The first extraction solution (4 M guanidine HC1) solubilized the polypeptides, mainly collagenous, not closely associated with the hydroxyapatite. The next extraction solution (4 M guanidine HC1, 0.5 M ethylenediaminetetraacedic acid (EDTA] solubilized the proteins more closely associated with the tooth mineral component. After extraction, the proteins were separated and isolated with gel electrophoresis. Protein molecular weights were determined and selected proteins were isolated for amino acid composition analysis. The two proteins isolated were tested for mammalian enamel protein antigenic determinants by a "Dot" immunobinding assay. The isolated proteins were enamelinlike by extraction criteria and amino acid composition. Further, the two proteins share antigenic determinants with mammalian enamel proteins.