用有限酶切法分析蛋白质的氨基酸序列

报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列.     

Relationship of bursal anti-steroidogenic peptide (BASP) and histone H1

Previous in vitro research from our laboratory has demonstrated the existence of a protein purified from the chicken bursa of Fabricius, with potent antisteroidogenic and antiproliferative action on granulose cells and lymphocytes, respectively called Bursal anti-steroidogenic peptide (BASP). This protein is heat-labile, basic, and amino- and carboxy-terminus blocked. In highly purified form, the protein presents as a doublet on SDS-PAGE electrophoresis with an apparent MW of approximately 29 and approximately 32 kDa. Recently, Nanoflow Q-TOF Mass Spectrometry amino acid sequencing allowed determination of a convincing partial amino acid sequence, strongly suggesting a probable relationship of BASP with histone H1. Bursal cDNA expression library screening, using an antibody produced against BASP, also identified a clone with a sequence matching histone H1. Presently, we have demonstrated that SDS-PAGE electrophoresis of highly purified and bioactive BASP, and commercially-available calf thymus derived histone H1, produced similar doublets at approximately the same apparent MW, and that the electrophoretic profile of these 2 preparations were strikingly similar following 2 dimensional gel electrophoresis. The BASP doublet produced on SDS-PAGE was recognized by a commercially available monoclonal antibody recognizing a highly conserved region of histone H1. Furthermore, calf thymus histone H1 was found to suppress mitogen-stimulated chicken B-cell proliferation in a concentration-related manner, similar to the action of BASP. These data indicate that BASP shares substantial structural homology with, and may be identical to, histone H1.     

The major outer membrane protein of Haemophilus ducreyi consists of two OmpA homologs.

The major outer membrane protein (MOMP) of Haemophilus ducreyi is an OmpA homolog that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as three species with apparent molecular weights ranging from 37,000 to 43,000. Monoclonal antibodies directed against this macromolecule were used to identify recombinant clones containing fragments of the gene encoding this protein. Nucleotide sequence analysis of these fragments confirmed that the MOMP encoded by the intact gene (momp) was a member of the OmpA family of outer membrane proteins. Construction of an isogenic H. ducreyi mutant unable to express the MOMP led to the discovery of a second outer membrane protein which migrated at the same rate on SDS-PAGE gels as the MOMP. N-terminal amino acid sequence analysis of this second protein revealed that its N terminus was nearly identical to that of the MOMP and also had homology with members of the OmpA family. Nucleotide sequence analysis of the region downstream from the momp gene revealed the presence of a partial open reading frame encoding a predicted OmpA-like protein. A modification of anchored PCR technology was used to obtain the nucleotide sequence of this downstream gene which was shown to encode a second OmpA homolog (OmpA2). The N-terminal amino acid sequence of OmpA2 was identical to that of the OmpA-like protein detected in the momp mutant. The H. ducreyi MOMP and OmpA2 proteins, which comigrated on SDS-PAGE gels and which were encoded by the tandem arranged momp and ompA2 genes, were 72% identical.     

Recombinant expression, purification and cross-reactivity of chenopod profilin: rChe a 2 as a good marker for profilin sensitization

Chenopod pollen is one of the major sources of allergens in some locations in the US, southern Europe and desert countries, and pollen profilin (Che a 2) is a major allergen. Recombinant Che a 2 (rChe a 2) has been produced in Escherichia coil cells with a final yield of 25 mg/l of cell culture. The expressed protein was isolated and structurally characterized by means of mass spectrometry, Edman degradation and circular dichroism. rChe a 2 displayed a molecular mass of 13 959 Da, which agrees with that of the amino acid sequence. The N-terminal amino acid sequence indicated the correct processing of the recombinant product. The immunological analysis of rChe a 2 showed IgG- and IgE-binding capabilities equivalent to those of its natural counterpart, Che a 2, isolated from the pollen. Inhibition experiments showed high cross-reactivity degrees with different allergenic sources. Inhibition degrees of >95% and >80% were obtained for chenopod profilin and, respectively, latex and pollen extracts, whereas 10-95% of inhibition was observed for different plant-derived foods. Due to its close relation to other allergenic profilins from pollens, plant-derived foods and latex, rChe a 2 could be a useful tool in clinical trials to detect profilin-allergic patients and perhaps, depending on its clinical relevance, in specific immunotherapy of these hypersensitive individuals.     

Precise and comparative pegylation analysis by microfluidics and mass spectrometry

Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.     

Molecular characterization of proteinaceous material in the Florida coastal Everglades

Dissolved organic nitrogen (DON) is the least known component of the nitrogen cycle, in part as a result of the lack of adequate analytical methods for its molecular characterization. In this study proteinaceous material in DON, collected at six geomorphologically different sites in the Florida coastal Everglades, was characterized by amino acid analysis and protein gel electrophoresis. The amino acid composition of the samples suggests that the canal DON was more degraded and subject to higher microbial inputs than the mangrove marshwater and marine end-member stations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) results supported this observation as distinctly different protein profiles were obtained for the canal waters compared to samples collected at other stations. These preliminary results highlight the potential of combining amino acid and intact protein analysis to fingerprint the sources of DON in different aquatic environments, and show SDS-PAGE as a potentially useful method to characterize DON.     

Physicochemical and immunochemical characterization of allergenic proteins from rye-grass (Lolium perenne) pollen prepared by a rapid and efficient purification method.

Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pI 5.0) and one basic (pI 9.0), termed 'R14a' and 'R14b' respectively, and R7 focused at pI 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data [Johnson & Marsh (1966) Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (R14a) and Rye III (R14b) from rye-grass pollen extract.     

Characterization of allergenic epitopes of Ory s1 protein from Oryza sativa and its homologs

Vaccination is the most effective technique suggested now days for allergy treatment. Recombinant-based approaches are mostly focused on genetic modification of allergens to produce molecules with reduced allergenic activity and conserved antigenicity. The molecules developed for vaccination in allergy possess significantly reduced allergenicity in terms of IgE binding, and therefore will not lead to anaphylactic reactions upon injection. This approach is probably feasible with every peptide allergen with known amino acid sequence. In this study an in silico approach was used to investigate allergenic protein sequences. Motif analysis of these sequences reveals the allergenic epitopes in the amino acid sequences. Physicochemical analysis of protein sequences shows that the homolog allergens of Ory s1 are highly correlated with the aromaticity, GRAVY and cysteine content. Moreover, phylogenetic analysis of Ory s1 with other sequences reveals that Oryza sativa japonica and Zea mays are close homologs, whilst Lolium perenne and Dactylis glomerata are found to be remote homologs. The multiple sequence alignment reveals of Ory s1 with all its homologs in this study reveals the high conservation of residues in DPBB_1 domain (amino acid residue positions 86- 164) and was found distinctly in all the sequences. These findings support the proposal that allergenic epitopes encompass conserved residues. The consensus allergenic was found to be mainly composed of hydrophobic residues. The functional sites of allergenic proteins reported in this study shall be attenuated to develop hypoallergenic vaccine. The sequence comparison strategy adopted in this study would pave way effective evolutionary analysis of these allergens.     

Hornet silk proteins in the cocoons produced by different Vespa species inhabiting Japan

We compared the components of hornet silk - a fibrous protein occurring in the cocoons produced by hornet larvae - among 6 species of the genus Vespa inhabiting Japan: V. simillima, V. dybowskii, V. crabro, V. mandarinia, V. ducalis, and V. analis. From the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal amino acid sequence analysis, and 5'-RACE, it was found that the major component proteins composing hornet silk could be divided into 6 groups. Among these 6 proteins, 5 proteins were common to the hornet silks of all 6 Vespa species. The SDS-PAGE major band corresponding to the remaining protein was observed only in the hornet silks of V. mandarinia and V. ducalis. This correspondence between V. mandarinia and V. ducalis can probably be explained in terms of the phylogenetic relationships of the Vespa species.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.     

Substrate Preference of γ-Glutamyltransferase from Caps of Fruiting Bodies of Lentinus edodes

Three kinds of proteins (BA-1, BA-2 and BA-3) allergenic to the IgE antibody of allergenic individuals were isolated from buckwheat seeds. These three proteins were essentially homogeneous as judged by both polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The amino acid composition of BA-1 and BA-2 was very similar, and the molecular weight of each allergenic protein was between 8000–9000 by SDS-polyacrylamide gel electrophoresis. One of them was a trypsin inhibitor, and their immunoreactivity was quite stable to heating at 100°C for 60 min.     

Identification of a new phospholipase D in Carica papaya latex

Phospholipase D (PLD) is a lipolytic enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. It catalyzes the hydrolysis and transphosphatidylation of glycerophospholipids at the terminal phosphodiester bond. The presence of a PLD in the latex of Carica papaya (CpPLD1) was demonstrated by transphosphatidylation of phosphatidylcholine (PtdCho) in the presence of 2% ethanol. Although the protein could not be purified to homogeneity due to its presence in high molecular mass aggregates, a protein band was separated by SDS-PAGE after SDS/chloroform-methanol/TCA-acetone extraction of the latex insoluble fraction. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by micro-LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (723 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 2424 bp encoding a protein of 808 amino acid residues, with a theoretical molecular mass of 92.05 kDa. From sequence analysis, CpPLD1 was identified as a PLD belonging to the plant phosphatidylcholine phosphatidohydrolase family.     

Capillary zone electrophoresis of insulin and growth hormone

The separation power of capillary zone electrophoresis was examined using highly purified and well-characterized biosynthetic human insulin, growth hormone, their derivatives, and related proteins. Mixtures of proteins were chosen to illustrate practical applications of this technique. Proteins differing slightly in structure, but equivalent in net charge, were not completely separated. Degradation of insulin by dilute acid treatment was followed by capillary zone electrophoresis, native polyacrylamide gel electrophoresis, and reversed-phase liquid chromatography. Excellent correlation was observed between these techniques. Simple equipment requirements and analysis times on the order of 10 min make capillary zone electrophoresis attractive for analytical protein separations.     

Predominant Low-Molecular-Weight Proteins in Isolated Brain Capillaries Are Histones

Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.     

Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.     

Recent progress in capillary electrophoretic analysis of amino acid enantiomers

This review highlights recent progresses in capillary electrophoresis (CE) analysis of amino acid enantiomers in the last decade. Various chiral selectors including cyclodextrins (CDs), bile salts, crown ethers, cinchona alkaloids, metal-chiral amino acid complexes, macrocyclic antibiotics and proteins have been employed to separate amino acid enantiomers. In the CE analysis of amino acids, the selection of the separation mode is one of the most important issues to obtain good resolution of target enantiomers. Among several separation modes, CD-modified capillary zone electrophoresis (CD-CZE), CD electrokinetic chromatography (CDEKC), micellar EKC (MEKC), CD-modified micellar electrokinetic chromatography (CD-MEKC), capillary electrochromatography (CEC), ligand-exchange CE (LE-CE), and nonaqueous CE (NACE) have been employed to the chiral analysis of amino acids. More than 160 published research articles collected from SciFinder Scholar databases from the year 2001 described the enantioseparations of amino acids by capillary-based electrophoresis. This review provides a comprehensive table listing the CE analysis of amino acid enantiomers with categorizing by the separation modes.     

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.     

Variant forms of galanin isolated from porcine brain

In a peptide concentrate, prepared from acid extracts of porcine brain, several galanin-like immunoreactive peptides were detected and two of these were purified. Characterization of the peptides by sequence analysis, mass spectrometry, and capillary zone electrophoresis identified them as a N-terminally nine residue elongated form of galanin, preprogalanin(24–61) amide, and as an N-terminally four residue truncated form of galanin corresponding to preprogalanin(37–61) amide. The former finding suggests that the removal of the signal peptide in preprogalanin occurs by enzymatic cleavage between glycine-23 and leucine-24. The presence of truncated galanin might refer to a mechanism, where galanin is inactivated by removal of functionally important amino acid residues from the N-terminus.