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PubMed1987

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Separation of isoforms of the glycoprotein hormones from human pituitary extracts.

Procedures for the high-resolution separation of microheterogeneous forms of the glycoprotein hormones, lutropin, follitropin and thyrotropin, present in human pituitary extracts are described. The sequential use of preparative isoelectric focusing and ion-exchange high-performance liquid chromatography (HPLC) allows the further discrimination of the isoforms of these glycoprotein hormones, previously considered homogeneous on the basis of their electrophoretic mobility. The ability of ion-exchange HPLC stationary phases to probe for slight differences in charge asymmetry expressed on the surface of these proteins raises the possibility of their rapid purification via the tandem on-line use of the same ion-exchange support with several different elution systems. Retention maps, illustrating the basis of this approach, are described.

Amino Acids↗

PubMed1992

Safety aspects in the manufacturing of plasma-derived coagulation factor concentrates.

Plasma-derived coagulation factor concentrates, prepared using traditional manufacturing processes, have transmitted viral diseases, especially AIDS, hepatitis B and hepatitis C to patients. To date, more extensive selection of blood donors, improved screening procedures of each plasma donation for direct and indirect viral markers, and newly developed virucidal procedures, especially pasteurization and solvent-detergent, together with extraction technologies of plasma proteins based on high-resolution chromatographic separations, have diminished considerably the risks of transmitting these pathogenic agents. To ensure safety, each production process must be carefully validated, following a rigorous scientific approach to assess its ability to inactivate or eliminate viruses. In addition, Good Manufacturing Practices must avoid any variation from these validated viral inactivation processes and must eliminate risks of potential downstream contamination of purified plasma fractions following viral inactivation or elimination steps. Other side-effects associated with conventional low-purity preparations, such as acute haemolytic anemia due to contamination by isohaemagglutinins, or immunosuppression possibly due to an overload in fibrinogen and immunoglobulins, have not been reported following infusion of highly purified coagulation factor concentrates. Present state-of-the-art virus inactivation and protein-purification technologies have significantly improved the safety of plasma coagulation factor concentrates.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Use of Mono Q high-resolution ion-exchange chromatography to obtain highly pure and active Escherichia coli RNA polymerase.

A method for the purification of highly pure and active Escherichia coli RNA polymerase holoenzyme is described. This method is simple, reproducible, and can be performed at room temperature. The procedure involves the high-performance liquid chromatography of a partially purified RNA polymerase sample on a Mono Q ion-exchange column. Under the conditions used, RNA polymerase holoenzyme is well separated from the core RNA polymerase and other impurities. The purified RNA polymerase contains virtually no impurities as judged by SDS-polyacrylamide gel electrophoresis. The purified RNA polymerase holoenzyme contains the sigma 70 subunit in stoichiometric amounts and is at least 90% active.

Anion Exchange Resins↗

PubMed1990

Purification of human smooth muscle filamin and characterization of structural domains and functional sites.

A method was developed to purify human smooth muscle filamin in high yield and structural domains were defined by using mild proteolysis to dissect the molecule into intermediate-sized peptides. Unique domains were defined and aligned by using high-resolution peptide mapping of iodinated peptides on cellulose plates. The amino- and carboxyl-terminal orientation of these domains within the molecule was determined by amino acid sequence analysis of several aligned peptides. In addition to the three unique domains which were identified, a number of smaller and larger fragments were also characterized and aligned within the intact molecule. These structural domains and related peptides provide a useful set of defined fragments for further elucidation of structure-function relationships. The two known functionally important binding sites of filamin, the self-association site and the actin-binding site, have been localized. Self-association of two monomers in a tail-to-tail orientation involves a small protease-sensitive region near the carboxyl terminal of the intact polypeptide chain. Sedimentation assays indicate that an actin-binding site is located near the blocked amino terminal of the filamin molecule. Sequences derived from large peptides mapping near the amino terminal show homology to the amino-terminal actin-binding site of alpha-actinin (chicken fibroblast and Dictyostelium), Dictyostelium 120-kDa actin gelation factor, beta-spectrin (human red cell and Drosophila), and human dystrophin. This homology is particularly interesting for two reasons. The functional form of filamin is single stranded, in contrast to alpha-actinin and spectrin which are antiparallel double-stranded actin cross-linkers. Also, no homology to the spectrin-like segments which comprise most of the mass of spectrin, alpha-actinin, and dystrophin was found. Instead, the sequence of a domain located near the center of the filamin molecule (tryptic 100-kDa peptide, T100) shows homology to the published internal repeats of the Dictyostelium 120-kDa actin gelation factor. On the basis of these results, a model of human smooth muscle filamin substructure is presented. Also, comparisons of human smooth muscle filamin, avian smooth muscle filamin, and human platelet filamin are reported.

Amino Acid Sequence↗

PubMed1994

Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase.

The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S. cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1980

Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA.

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.

Animals↗

PubMed1986

Purification and structural analysis of pyocyanin and 1-hydroxyphenazine.

Pyocyanin and related members of the phenazine family are produced by Pseudomonas aeruginosa and have been associated with events of pathophysiological importance. Pyocyanin and its base hydrolysis product 1-hydroxyphenazine were purified to homogeneity by reverse-phase high-pressure liquid chromatography. Their mass spectrometric behaviour was examined with a view to evaluating the use of high-resolution chromatography/mass spectrometry in studying phenazine-mediated effects in man. The molecular mass of naturally derived pyocyanin was determined as 210 Da by thermospray liquid chromatography/mass spectrometry and confirmed by desorption electron-impact mass spectrometry. Mass spectrometric data could not be obtained by fast-atom bombardment or desorption chemical ionisation, techniques commonly used to determine molecular mass of polar or thermally labile species. The thermal lability of underivatised pyocyanin precluded analysis by gas chromatography/mass spectrometry. In contrast to pyocyanin, mass spectrometric data were readily obtained for 1-hydroxyphenazine, using direct probe analysis as well as with gas and liquid chromatography inlet systems.

Chromatography, Gas↗

PubMed1987

Monitoring the purification by high-performance liquid chromatography of cardiotoxins from Naja mossambica mossambica using phase-sensitive two-dimensional nuclear magnetic resonance.

High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

Amino Acids↗

PubMed1985

Identification of transformation products arising from bacterial oxidation of codeine by Streptomyces griseus.

14-Hydroxycodeine and norcodeine were rigorously identified as products arising from codeine oxidation by Streptomyces griseus ATCC 10137. Both products were routinely detected in extracted culture filtrates after growth of cells in the presence of codeine for 1 week. Under these conditions, about 4 mol% of the codeine starting material was consumed, with norcodeine and 14-hydroxycodeine representing the only identifiable transformation products (molar ratio, 4:1, respectively). Extraction of a series of culture filtrates and purification of the pooled metabolites by thin-layer and high-pressure liquid chromatography led to the isolation of both biological products, the structures of which were verified by high-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. The identities of both biological products were further confirmed by comparison of their spectral properties with those of authentic standards. This is the first report providing structural evidence for the biological formation of 14-hydroxycodeine from codeine and of codeine oxidation by S. griseus.

Biotransformation↗

PubMed1996

High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.

Animals↗

PubMed2026

Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.

Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.

DNA extraction↗

PubMed1989

Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen.

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Animals↗

PubMed1991

Purification of secreted recombinant proteins from Escherichia coli.

Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic compartments. Thus, hGH and muIL-2 extracted from the cytoplasm of E. coli have been characterized as high molecular weight disulfide-bonded oligomers. It is likely that oligomerization occurs as the polypeptides are released from the reducing environment of the cytoplasm. In contrast, secreted hGH and muIL-2 extracted from the periplasm of E. coli by osmotic shock displayed the properties of a property folded native protein with correct disulfide pairing. In the case of muIL-2 only a small residual fraction (approximately 15%) of the purified secreted protein exhibited incomplete oxidation of cysteine (35). Secretion of heterologous proteins into the periplasm prevents their exposure to the action of proteases located in the cytoplasm of E. coli (58). The smaller polypeptides such as somatostatin (59), IGF-1 (46), and hEGF (54) are known to be particularly susceptible to intracellular degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.

Amino Acid Sequence↗

PubMed1992

Co-purification of p34cdc2/p58cyclin A proline-directed protein kinase and the retinoblastoma tumor susceptibility gene product: interaction of an oncogenic serine/threonine protein kinase with a tumor-suppressor protein.

Proline-directed protein kinase (PDPK) is characterized as a cytoplasmic oncogenic serine/threonine kinase that is activated by growth factor-mediated mechanisms and is proposed to function in mammalian somatic cells as an S phase promoting factor. The present study was undertaken to assess the hypothesis that p34cdc2/p58cyclinA PDPK is a physiologically relevant form of the p34cdc2 protein kinase that phosphorylates and inactivates the product of the retinoblastoma/osteosarcoma tumor susceptibility gene (Rb protein). In the course of these studies it was determined (fortuitously) that the p34cdc2/p58cyclinA PDPK purified from the cytosol of FM3A mouse mammary carcinoma cells was 'contaminated' by several high molecular weight substrate proteins that essentially co-purified with the protein kinase, one of which was identified as the Rb protein itself (p105Rb). High-resolution fast protein liquid chromatography (FPLC) revealed that the Rb protein co-purified with a particular subset of the PDPK heterodimer, i.e. with a single species of the 58 kDa cyclinA doublet. The subset of PDPK associated with the Rb protein exhibited somewhat lower specific enzyme activity, as judged by in vitro kinase assays and comparative Western blotting. Immunoprecipitation studies confirmed that p105Rb is physically associated with the p34cdc2/p58cyclin A PDPK. Further studies confirmed that the underphosphorylated Rb protein (p105Rb) present in G1 lysates of synchronized human MG63 osteosarcoma cells could be readily phosphorylated by purified PDPK in vitro, resulting in the characteristic shift in the apparent molecular mass (SDS-PAGE) of the Rb protein that is reported to accompany the hyperphosphorylation and functional inactivation of this protein. Moreover, the induction of the cyclin A subunit of PDPK in these synchronized MG63 cells was found to be closely correlated with the cell cycle-dependent phosphorylation of the Rb protein. From these studies it is concluded that the growth factor-sensitive PDPK is a physiological Rb kinase, which may function to inactivate the Rb protein in vivo.

Animals↗

PubMed1992

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.

Biotechnology↗

PubMed1994

High-resolution chromatography of proteins in short columns and adsorptive membranes.

Short chromatographic columns prepared from stacks of microporous adsorptive membranes are promising for preparative-scale fractionation of even rather closely related proteins, but careful selection of operating conditions is needed for success. It has been shown that existing devices exhibit very low internal diffusional resistance, and that resolution is almost totally independent of percolation velocity. Total column length is short, however, and the number of plates exhibited under isocratic low-loading conditions is small, on the order of 100. Simulations using a large survey of protein thermodynamic data show that one can frequently obtain excellent protein separations in these short columns by using the sensitivity of protein adsorption equilibria to eluting solvent composition. In fact, some proteins can be separated in a single stage utilizing this 'on-off' behavior and properly selected solvent gradients. Solely on-off, or differential elution, behavior cannot often be depended upon under the mild conditions need for preparative operations. Carefully programmed gradient elution can frequently produce acceptable purification by maximizing both differential elution and differential migration, resulting in protein separations in columns of less than fifty plates. Means for doing this are described for some simple situations, and criteria are provided for selecting modulator gradient schedules.

Animals↗

PubMed1996

High-level bacterial expression of mitochondrial transport proteins.

The inability to obtain abundant quantities of purified, functional membrane proteins has represented a significant impediment to the goal of obtaining high-resolution structural information. In this review, procedures are described which have been developed in this laboratory and enable the high-level bacterial expression and subsequent purification of functional mitochondrial citrate transport proteins from yeast and rat liver. The data that we have obtained using these procedures and related results from other laboratories are discussed. Additionally, the general applicability of this approach to most mitochondrial transport proteins as well as to other types of membrane proteins is considered. Finally, relevant considerations when contemplating the use of this methodology and the likely value of this approach in future areas of research are explored.

Animals↗