A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.     

N-terminal sequence similarities between components of the multicatalytic proteinase complex

The multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase which is composed of many different types of subunit. As part of a study of the possible relationships between subunits, polypeptides derived from the multicatalytic proteinase from rat liver have been subjected to N-terminal amino acid sequence analysis. Although several of the subunits are blocked at their N-termini, sequences have been obtained for 7 of the polypeptides. Each of the 7 sequences is unique but they show considerable sequence similarity, suggesting that the proteins are encoded by members of the same gene family.     

Assignment of a polymorphic polypeptide to the human mitochondrial DNA unidentified reading frame 3 gene by a new peptide mapping strategy

A new method of peptide analysis is presented which allows assignment of unknown proteins to coding regions of genomes which have been sequenced. This approach involves comparison of the molecular weights of peptides generated by partial proteolytic digestion with those predicted for a protein whose primary amino acid sequence is deduced from a corresponding nucleotide sequence. The proteolytic digestions are accomplished in situ in the stacking gel of a two-dimensional polyacrylamide gel system. We have used this system to show that two variant proteins of the human mitochondrial DNA, MV-1 and MV-2, are allelic and encoded by the unidentified reading frame 3 (URF 3) gene. This assignment was supported by sequence analysis of a clone of this mtDNA region from a HeLa cell line which expresses the uncommon variant MV-2. Four nucleotide changes were found in HeLa URF 3, relative to the reported sequence from human placenta. Two of these changes alter the primary amino acid sequence of the encoded protein. It is proposed that one of those amino acid changes may account for the observed molecular weight variation in MV-1 and MV-2 by proteolytic cleavage, conformational change, or secondary modification. We have used this method to also assign a mitochondrially translated protein to URF 6. These are the first assignments of mitochondrially synthesized polypeptides to human URF genes and prove conclusively that at least some of these genes are expressed in human cells.     

Primary structure of a human small nuclear ribonucleoprotein polypeptide as deduced by cDNA analysis

Anti-Sm is an antibody specificity often associated with the autoimmune disease systemic lupus erythematosus. The polypeptides Sm-B'/B (estimated molecular mass 27 and 26 kDa, respectively) are primary targets of Sm antibodies. Sm-B'/B are part of the core polypeptides of small ribonucleoprotein particles (snRNP) involved in pre-mRNA splicing. Sm-B'/B share the same amino-terminal sequence as we determined by microsequence analyses of the purified polypeptides. Oligonucleotide probes based on that sequence were used to isolate seven clones from a human lymphoblastoid cDNA library in lambda gt10. The clones contained a single coding region for a protein of approximately 25 kDa. The predicted amino-terminal sequence was identical to that of the isolated Sm-B'/B polypeptides. In vitro translation experiments produced a protein immunoreactive with human polyclonal anti-Sm antibodies. The isolation of only one unique cDNA sequence suggests that Sm-B'/B may be post-translational variants encoded by a single message. The specific structural features which distinguish Sm-B' from Sm-B have yet to be determined. Northern blot analysis confirmed the diverse tissue and species distribution expected for these immunologically conserved polypeptides. The Sm-B'/B primary sequence is rich in proline (20%) and glycine (15%) residues. The prolines are concentrated in the carboxyl-terminal half of the protein and display a repetitive unit that is shared with other snRNP and nucleic acid binding proteins. Analysis of these arrays suggests an eight residue proline-rich consensus sequence with potential as either an RNA binding domain, or as a site of protein/protein interaction.     

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus

Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically cleaved at glutaminyl-methionine and glutaminyl-glycine, respectively, to produce the two viral capsid proteins.     

Microtubule affinity chromatography: a new technique for isolating microtubule-binding proteins from rat pancreas.

We have developed an affinity chromatography method for the isolation of microtubule-associated proteins (MAPs) from soluble cytoplasmic extracts of rat pancreas. Among the ten proteins which copurify with pancreas tubulin on a colchicine derivatives-affinity chromatography, three polypeptides of respectively 58, 55 and 48 kDa strongly bind to the microtubule affinity column. To begin to characterize these proteins, we have generated polyclonal antibodies against tau polypeptides from brains of immature chicken or rat. As judged by immunoblots, the three polypeptides seem to be immunologically related to the tau proteins previously localized in brain.     

Evolution of the tryptophan synthetase of fungi. Analysis of experimentally fused Escherichia coli tryptophan synthetase alpha and beta chains

During evolution of fungi, the separate tryptophan synthetase alpha and beta polypeptides of bacteria appear to have been fused in the order alpha-beta rather than the beta-alpha order that would be predicted from the order of the corresponding structural genes in all bacteria. We have fused the tryptophan synthetase polypeptides of Escherichia coli in both orders, alpha-beta and beta-alpha, with and without a short connecting (con) sequence, to explore possible explanations for the domain arrangement in fungi. We find that proteins composed of any of the four fused polypeptides, beta-alpha, beta-con-alpha, alpha-beta, and alpha-con-beta, are highly active enzymatically. However, only the alpha-beta and alpha-con-beta proteins are as active as the wild type enzyme. All four fusion proteins appear to be less soluble in vivo than the wild type enzyme; this abnormal characteristic is minimal for the alpha-con-beta enzyme. The alpha and beta domains of the four fusion polypeptides were not appreciably more heat labile than the wild type polypeptides. Competition experiments with mutant tryptophan synthetase alpha protein, and the fusion proteins suggest that in each fusion protein the joined alpha and beta domains have a functional tunnel connecting their alpha and beta active sites. Three tryptophan synthetase beta'-alpha fusion proteins were examined in which the carboxyl-terminal segment of the wild type beta polypeptide was deleted and replaced by a shorter, unnatural sequence. The resulting deletion fusion proteins were enzymatically inactive and were found predominantly in the cell debris. Evaluation of our findings in relation to the three-dimensional structure of the tryptophan synthetase enzyme complex of Salmonella typhimurium (5) and the results of mutational analyses with E. coli suggest that tryptophan synthetase may have evolved via an alpha-beta rather than a beta-alpha fusion because in beta-alpha fusions the amino-terminal helix of the alpha chain cannot assume the conformation required for optimal enzymatic activity.     

动物源活性蛋白多肽的分离纯化方法研究进展

动物类中药的有效成分以蛋白多肽为主,因此活性蛋白多肽具有重要的医疗保健价值。文章分析了沉淀法、色谱法、膜分离法以及电泳法的基本原理和主要适用范围,综述了这些方法在动物源活性蛋白多肽的分离纯化中的应用,为动物源蛋白多肽的分离纯化与进一步研究提供参考,以期开发出高效、经济和环保的蛋白多肽分离纯化新技术。     

A theoretical method for distinguishing between soluble and membrane proteins

A method for distinguishing between membrane and soluble proteins in an amino acid sequence was developed, using only two parameters associated with the hydrophobicity: the average hydrophobicity and the power spectral density of period longer than 30 residues. The power spectral density was calculated by a maximum entropy method of Fourier transformation. Membrane proteins could be distinguished from soluble proteins with a distinction rate as high as 97%. This fact strongly suggests that the morphology of proteins, i.e., membrane or soluble forms, is determined thermodynamically through the hydrophobicity of polypeptides.     

Surface recognition elements of membrane protein oligomerization

Although certain membrane proteins are functional as monomeric polypeptides, others must assemble into oligomers to carry out their biological roles. High-resolution membrane protein structures provide a valuable resource for examining the sequence features that facilitate-or preclude-assembly of membrane protein monomers into multimeric structures. Here we have utilized a data set of 28 high-resolution alpha-helical membrane protein structures comprising 32 nonredundant polypeptides to address this issue. The lipid-exposed surfaces of membrane proteins that have reached their fully assembled and functional biological units have been compared with those of the individual subunits that build quaternary structures. Though the overall amino acid composition of each set of surfaces is similar, a key distinction-the distribution of small-xxx-small motifs-delineates subunits from membrane proteins that have reached a functioning oligomeric state. Quaternary structure formation may therefore be dictated by small-xxx-small motifs that are not satisfied by intrachain contacts.     

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.     

Microtubule-binding domain of tau proteins

Limited chymotryptic digestion of whole tau proteins produced a fragment of Mr 14,000 (CT14), which was able to bind to microtubules reconstituted from tubulin alone in the presence of taxol. This fragment was also found to persist in microtubules when microtubules consisting of tau proteins and tubulin were digested by chymotrypsin. Analysis of amino acid composition revealed that CT14 was rich in lysine and proline residues, suggesting unique structure of microtubule-binding domain of tau proteins. Amino-terminal sequence of CT14 was determined to be Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr-Pro-Gly-Ser-Arg-Ser-Arg-X-Pro-Ser-Leu-Pr o. No heterogeneity was detected in this amino-terminal sequence of 19 residues. Five species of polypeptides consisting of tau proteins were separated from each other by gel electrophoresis and subjected to chymotryptic digestion. CT14 was produced from each of the tau polypeptides by chymotryptic digestion, indicating that all tau polypeptides have a common microtubule-binding domain.     

Reducing protein concentration range of biological samples using solid-phase ligand libraries

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.     

Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2.

The biochemical processes responsible for the recognition and repair of cisplatin-damaged DNA in human cells are not well understood. We have developed a damaged DNA affinity precipitation technique that allows the direct visualization and characterization of cellular proteins that bind to cisplatin-damaged DNA. The method separates damaged DNA-binding proteins from complex radiolabeled cell mixtures and further resolves them into individual polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This technique is complementary to gel retardation and Southwestern blotting analyses that have been previously used to identify cellular components that specifically bind to cisplatin-damaged DNA. Using this technique, we have characterized a set of HeLaS3 nuclear proteins of 26.5, 28, 90, and 97 kDa that specifically bind to cisplatin-DNA adducts. Competition studies with soluble cisplatin-damaged DNA confirmed these findings. The major cisplatin-damaged DNA-binding proteins of 26.5 and 28 kDa recognized adducts of DNA modified with cisplatin but not with its trans-isomer or with UV radiation. These proteins were purified 450-fold to near homogeneity by ion-exchange and cisplatin-damaged DNA affinity chromatography. Amino-terminal sequence analysis showed that the 26.5- and 28-kDa proteins were identical to high mobility group (HMG) proteins HMG-2 and HMG-1, respectively.     

N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

Characterization of two platelet aggregation inhibitor-like polypeptides from viper venom

Two polypeptides, eristocophins I and II, have been characterized from leaf-nosed viper (Eristocophis macmahoni) venom. They contain 10 half-Cys residues of a total of 61/62 residues, have 72% residue identity, and exhibit similarities to platelet aggregation inhibitors and segments of adhesive proteins. Eristocophin I contains the sequence Arg-Gly-Asp, known to inhibit fibrinogen interaction with the platelet receptor. Eristocophin II has Met instead of Arg in this sequence, and an adjacent Trp-Asn-Asp segment. The latter is also typical of adhesive proteins, thus linking two potentially functional segments in one molecule. Exchanges are maximal in these segments, suggesting that the polypeptides exhibit functional divergence with isoform differences in important regions.     

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.     

Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein

Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen-like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4-hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH-terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N-acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein.