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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF). In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography. After expression in E. coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization. The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase. The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP. In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis. The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins. Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD. This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process. Remaining DNA and HU protein were separated by Heparin HyperD. The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml. The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample. In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs.
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PMID:Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli. 1068 Oct 38

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.
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PMID:Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli. 1069 29

The present study reports a procedure developed for the identification of SDS-polyacrylamide gel electrophoretically separated proteins using an electrospray ionization quadrupole time-of-flight mass spectrometer (Q-TOF MS) equipped with pressurized sample introduction. It is based on in-gel digestion of the proteins without previous reduction/alkylation and on the capability of the Q-TOF MS to provide data suitable for peptide mass fingerprinting database searches and for tandem mass spectrometry (MS/MS) database searches (sequence tags). Omitting the reduction/alkylation step reduces sample contamination and sample loss, resulting in increased sensitivity. Omitting this step can leave disulfide-connected peptides in the analyte that can lead to misleading or ambiguous results from the peptide mass fingerprinting database search. This uncertainty, however, is overcome by MS/MS analysis of the peptides. Furthermore, the two complementary MS approaches increase the accuracy of the assignment of the unknown protein. This procedure is thus, highly sensitive, accurate, and rapid. In combination with pressurized nanospray sample introduction, it is suitable for automated sample handling. Here, we apply this approach to identify protein contaminants observed during the purification of the yeast DNA mismatch repair protein Mlh 1.
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PMID:Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. 1074 Aug 54

Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.
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PMID:Mechanism-based inactivation of cytochrome P450 3A4 by L-754,394. 1075 76

From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, alpha and beta, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine-HCl, erinacin dissociates into alpha-subunits and beta-subunit decamers. From these results the subunit assembling of erinacin has been formulated as alpha(10).2beta(10). The molecular weight of the subunits and of the beta-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.
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PMID:The antihemorrhagic factor, erinacin, from the European hedgehog (Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains. 1077 56

Virosomes are cytoplasmic sites of replication of vaccinia virus DNA and were prepared from virus-infected HeLa cells. The early virosomal proteins were 35S-labelled and SDS polyacrylamide gel electrophoresis revealed the presence of three major early 35S-labelled proteins of 34, 24 and 45 kDa. The masses of molecules present in the 34 and 24 kDa proteins were measured by the convenient and sensitive MALDI TOF mass spectroscopy technique. Identification of the three virosomal proteins was carried out by MALDI mass spectroscopy of corresponding tryptic digests. For each protein at least 13 measured masses matched, within less than 0.1 Da, calculated tryptic peptides of the vaccinia virus proteins H5R (34 kDa), E3L (24 kDa) and E5R (45 kDa). In addition, virosomes contained several structural proteins from the infecting virus and a 45 kDa keratin-related protein. This work demonstrates directly that the abundant early vaccinia virus proteins H5R, E3L and E5R are associated with the virosomes.
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PMID:Identification by mass spectroscopy of three major early proteins associated with virosomes in vaccinia virus-infected cells. 1085 61

Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS-proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive electroelution steps were required to obviate pH changes in the electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 microg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single electroelution step; (ii) using electroelution from a single gel of 0.3-cm(2) cross-sectional area; and (iii) using a protein load of 2 (in one case 4) microg. However, the migration rates of the Cascade blue-SDS-protein-barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.
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PMID:Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis. 1096 11

A cytosolic alanyl aminopeptidase (AAP-S) was purified to homogeneity from human liver cytosol. The molecular weight of the purified enzyme was calculated to be approximately 98,000 on TOF-MS and 90,000 on SDS-PAGE in the presence of beta-ME. These findings suggest that the enzyme exists as a monomeric form in human liver cytosol. The enzyme rapidly hydrolyzed the substrates Ala-, Lys- and Phe-MCAs, and moderately hydrolyzed Met-, Leu-, Tyr- and Lys-Ala-MCAs at pH ranging from 7.5 to 8.0. The order of the K(cat)/K(m) values of AAP-S at the optimal pH was Arg->Arg-Arg->Met->Leu->Lys->Phe->Lys-Ala->Tyr->Ala-MCAs. It was strongly inhibited by bestatin, leuhistin, actinonin, amastatin, 1, 10-phenanthroline, DFP, PCMBS, Zn(2+), Cd(2+), Co(2+), Cu((2+)), Hg(2+) and puromycin. AAP-S was approximately 80 times more sensitive than human seminal plasma AAP (aminopeptidase N, membrane type). The amino acid sequence of the first 60 residues of AAP-S was highly homologous with the N-terminal amino acid sequence of the rat liver puromycin-sensitive enkephalin-degrading aminopeptidase. These physicochemical properties and findings indicate that AAP-S from human liver cytosol is identical to those of other puromycin-sensitive aminopeptidase(s). Furthermore, with immunohistochemistry the enzyme was strongly stained in the cytoplasm of liver cells and renal tubules, and was ubiquitously localized in various human tissues.
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PMID:Puromycin-sensitive alanyl aminopeptidase from human liver cytosol: purification and characterization. 1097 16

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS, and 82000 on non-denatured PAGE and 82000 on SDS-PAGE in the absence or presence of beta-ME. These findings suggest that the enzyme assumes a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg-MCA and moderately hydrolyzed Ala-Arg-MCA in a pH range of 7. 5 to 9.5. The K(in), K(cat) and K(cat)/K(m) values of DPP III at optimal pH (pH 8.5) were 290 microM, 18.0 s(-1) and 6.21x10(4) s(-1)M(-1) for Arg-Arg-MCA and 125 microM, 4.53 s(-1) and 3.62x10(4) s(-1)M(-1) for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS, NEM, beta-ME and iodoacetamide. Furthermore, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG, and we determined the cDNA structure and deduced the amino acid sequence. The cDNA designated as lambdaRDIII-11 is composed of 2640 bp of nucleotides in length and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH in structure, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. These findings suggest that DPP III is a metalloprotease that is probably regulated by SH modification. The DPP III antigen was extensively detected in the cytosol of various rat tissues by the immunohistochemical examination of the protein.
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PMID:Molecular cloning and immunohistochemical localization of rat dipeptidyl peptidase III. 1097 17

A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.
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PMID:Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme. 1103 47


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