Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The preparation and optimization of a new monolithic chymotrypsin bioreactor for online protein digestion is described. Silica monolithic supports have been activated with epoxide functionalities following an optimized in situ procedure and used for covalent immobilization of chymotrypsin in one-step reaction under different conditions. A total of four bioreactors were prepared and characterized in terms of the amount of immobilized enzyme and apparent active units by using a standard substrate, N-benzoyl-L-tyrosine p-nitroanilide (BTPNA). The stability of the bioreactors was evaluated and the morphology of the support after immobilization and use was studied by SEM analysis. The proteolytic activity of the optimized chymotrypsin bioreactor was evaluated using HSA as a model protein by online coupling of the bioreactor with LC-ESI-MS. With the online protocol, complete protein digestion in 120 min was achieved with a sequence coverage of 97.3%.
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PMID:Chymotrypsin immobilization on epoxy monolithic silica columns: development and characterization of a bioreactor for protein digestion. 1792 85

An artificial protein containing alternating hydrophilic-hydrophobic blocks of amino acids was designed in order to mimic the structure of synthetic multiblock copolymers. The hydrophobic block consisted of the six amino acids Ala Ile Leu Leu Ile Ile (AILLII) and the hydrophilic block of the eight amino acids Thr Ser Glu Asp Asp Asn Asn Gln (TSEDDNNQ). The coding DNA sequence of the cluster was inserted into an commercial pET 30a(+) vector using a two step strategy. The expression of the artificial protein in Escherichia coli was optimized using a temperature shift strategy. Only at cultivation temperature of 24 degrees C after induction expression was observed, whereas at 30 and 37 degrees C no target protein could be detected. Cells obtained from a 15L bioreactor cultivation of E. coli were disintegrated by mechanical methods. Interestingly, glass bead milling and high pressure homogenization resulted in a different solubility of the target protein. The further purification was carried out by affinity chromatography using the soluble homogenized protein. Extreme conditions (6M urea, 0.5M NaCl) were applied in order to prevent aggregation to insoluble particles. The designer protein showed an extremely high tendency to form dimers or trimers caused by intermolecular interactions which were even not broken under the conditions of SDS-polyacrylamide gel electrophoresis, rendering the behavior during purification different from proteins usually found in nature. The protein preparation was not completely pure according to SDS-PAGE stained by Coomassie blue or silver. In MALDI-TOF-MS, nano-ESI qTOF-MS of the entire protein preparation and nano-ESI-MS after digestion by trypsin and chymotrypsin impurities were not detectable.
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PMID:Cloning, expression, purification, and characterization of a designer protein with repetitive sequences. 1835 48

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.
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PMID:[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry]. 1881 79

The development of a new mixed bioreactor for proteomic studies based on trypsin and chymotrypsin is described. Trypsin and chymotrypsin were simultaneously bonded to an epoxy monolithic silica column (100 mmx4.6 mm id) in a one-step reaction via epoxy-groups. In order to compare the catalytic properties of the two enzymes in the isolated and in the multi-enzymatic approach, two other single enzyme bioreactors based on trypsin and chymotrypsin were prepared following the same immobilization protocol. The kinetic parameters of the multi-enzymatic bioreactor were derived and it was demonstrated that it retains the individual catalytic activity of the two enzymes. To prove the power of this experimental approach the new mixed bioreactor was integrated in an LC-ESI-MS/MS system for digestion, enrichment, separation and identification of the test protein insulin-like growth factor binding-protein 1 (IGFBP-1). The peptide map and protein sequence coverage obtained with the three bioreactors were compared. The results clearly indicate that the proposed multi-enzyme approach can reduce both digestion and analysis time, accelerate data interpretation and increase the confidence degree in protein identification.
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PMID:On-line multi-enzymatic approach for improved sequence coverage in protein analysis. 1930 25

A new methodology for the detection and isolation of serine proteases in complex mixtures has been developed. It combines the characterization of crude samples by electrospray tandem mass spectrometry (ESI-MS/MS) in a multi-substrate assay and the differentiated sensitive detection of the responsible enzymes by means of liquid chromatography hyphenated online to biochemical detection (BCD). First, active samples are identified in the multi-substrate assay monitoring the conversion of eight substrates in multiple reaction monitoring in parallel within 60s. Hereby, the product patterns are investigated and the suitable peptide as substrate for BCD analysis is selected. Subsequently, the active proteases are identified online in the continuous-flow reactor serving as BCD after non-denaturing separation by size-exclusion chromatography and ion-exchange chromatography. For BCD, the selected para-nitroaniline (pNA) labeled peptide is added post-column and is cleaved by eluting proteases under release of the coloured pNA in a reaction coil (reaction time 5min). The method was optimized and the figures of merit were characterized with trypsin and chymotrypsin serving as the model proteases. For trypsin, a limit of detection in LC-BCD of 0.1U/mL corresponding to an injected amount of 0.4ng protein ( approximately 18fmol) was observed. The BCD signal remained linear for an injected enzyme concentration of 0.3-10U/mL (1.3-42ng enzyme). The method was applied to the characterization of the crude venom of the pit viper Bothrops moojeni and the extracellular protease of the pathogenic amoeba Acanthamoeba castellanii. In the two samples, fractions with proteolytic activity potentially interfering with the blood coagulation cascade were identified. The described methodology represents a tool for serine protease screening in complex mixtures by a fast ESI-MS/MS identification of active samples followed by the separation and isolation of active sample constituents in LC-BCD.
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PMID:Development of a liquid chromatography-based screening methodology for proteolytic enzyme activity. 1934 51

Granulocyte-colony stimulating factor stimulates production and antibacterial function of neutrophiles. Therapy using the recombinant protein drug represents a major step forward in oncology. The protein has not been, however, completely sequenced at the protein level and this formed the rationale of the current study. Recombinant G-CSF (filgrastim) was run on two-dimensional gel electrophoresis (2DE), the protein was in-gel digested with trypsin and chymotrypsin, and peptides were analysed on Nano-ESI-LC-MS/MS (high performance ion trap, HCT). Bioinformatic tools used were Mascot v2.2 and Modiro(TM) v1.1 softwares. A single spot was detected on 2DE and peptides resulting from in-gel digestion were unambiguously identified by the MS/MS approach leading to complete sequencing when both searching engines were applied. N-terminal methionine loss, N-terminal methionine oxidation and amidination were observed. Both softwares identified modifications. Complete sequencing by a non-sophisticated and rapid gel-based mass spectrometry approach confirmed the primary structure predicted from nucleic acid sequences. A chemical modification of glutamine 26 with the interim name PentylamineBiotin (Unimod accession number #800) compatible with biotinylation with 5-(biotinamido) pentylamine by the producer was detected by both softwares. Although there is some evidence that biotinylated G-CSF analogues are active, it remains open whether this modification may be responsible for the side effects observed or lead to changes of antigenicity.
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PMID:Complete sequencing of the recombinant granulocyte-colony stimulating factor (filgrastim) and detection of biotinylation by mass spectrometry. 1952 10

Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.
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PMID:Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry. 1954

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotein nm23, crustacyanin-C1 and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.
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PMID:Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress. 1957 23

Knowledge on complete sequences is pivotal to identify splice variants, generate specific antibodies and predict conformation. A simple analytical approach to obtain 100% sequence coverage, however, is currently not available. Recombinant gamma-aminobutyric acid A receptor subunits were from insect SF9 cells that were co-transfected with rat alpha1 and His-tag beta 3. The complex of these two subunits was run on blue-native PAGE, followed by multidimensional electrophoretic steps. Spots resolved at the third electrophoretic step were in-gel digested with trypsin, chymotrypsin and Asp-N. In-gel modification of lysines by acetylation was carried out to increase sequence coverage. Subsequently, peptides were analyzed by nano-ESI-LC-MS/MS using both, collision-induced dissociation and electron transfer dissociation principles. When results from trypsin, chymotrypsin and Asp-N digestion were combined, a single peptide [424 KKTHLRRRSSQL 435] was still not identified. In-gel lysine acetylation leads to unambiguous identification of this peptide by the use of MASCOT v2.2. The use of the Modiro software along with MASCOT, however, was able to provide 100% sequence coverage even without the use of in-gel lysine acetylation. It was observed that the use of trypsin, chymotrypsin and Asp-N with bioinformatic handling by the MASCOT and Modiro software is sufficient to obtain complete sequencing of a highly hydrophobic membrane protein.
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PMID:Complete sequencing of GABAA receptor subunit beta 3 by a rapid technique following in-gel digestion of the protein. 1958 15

The analysis of highly hydrophobic proteins is still an analytical challenge. Using a recombinant gamma-aminobutyric acid A (GABAA)-receptor subunit as a model protein, we developed a gel-based proteomic approach for high MS/MS-peptide sequence coverage identification. Protein samples were separated by multi-dimensional gel electrophoresis and the three protein spots representing the GABAA-receptor subunit alpha-1 from the last electrophoretic step were used for in-gel digestion with trypsin, chymotrypsin and subtilisin, followed by subsequent mass-spectrometric identification by nano-ESI-LC-MS/MS Qstar XL (quadrupole time-of-flight (qQTOF)) and linear ion trap (LIT) LTQ XL identification. This protocol allows the unambiguous identification of the GABAA-receptor alpha-1 subunit protein with 100% sequence coverage, thus covering all four hydrophobic transmembrane domains. This protocol differs from other methods in the selection of enzymes, digestion conditions and use of the two mass spectrometry principles. The protocol takes approximately 10 d to complete and may represent a step forward in the complex analysis of other membrane or hydrophobic proteins.
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PMID:Gel-based mass spectrometric analysis of a strongly hydrophobic GABAA-receptor subunit containing four transmembrane domains. 1970 90


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