共查询到20条相似文献,搜索用时 46 毫秒
1.
A. A. Kaloshin E. V. Gatypova N. A. Mikhailova 《Applied Biochemistry and Microbiology》2011,47(8):780-788
Two recombinant forms of the outer membrane protein F (OprF) of Pseudomonas aeruginosa were obtained, the full-length protein OprF and the C-terminal part of the OprF protein (aa 192–342). As a result of double
immunizations, these recombinant proteins provided mice with resistance to experimental intraperitoneal challenge with P. aeruginosa. The best protective effects were observed at a dose of 25 μg for OprF and 50 μg for the truncated OprF variant (indices
of efficiency were 3.3 and 2.8, respectively). Rabbit antisera immune to the recombinant proteins were also able to protect
mice from the experimental infection with P. aeruginosa. Indices of efficiency were 6.4 for OprF and 6.0 for the OprF C-terminal part; these values were approximately two times
as high as the effect of sera from intact test animals (3.2). 相似文献
2.
Photobacterium damsela α2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16–497 aa or 113–497 aa). Expression and activity assays indicated that
the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated
forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16–497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST(N),
the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5–10.0. A divalent metal ion was not required for the sialyltransferase
activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly.
Mingchi Sun and Yanhong Li contributed equally to this work. 相似文献
3.
Xiao-Hong Guo Jing Jiang Bai-Chen Wang Hui-Yu Li Yu-Cheng Wang Chuan-Ping Yang Gui-Feng Liu 《Molecular biology reports》2010,37(3):1183-1190
The ThPOD1 gene encodes a peroxidase and was isolated from a Tamarix hispida NaCl-stress root cDNA library. We found that ThPOD1 expression could be induced by abiotic stresses such as cold, salt, drought and exogenous abscisic acid. These findings suggested
that ThPOD1 might be involved in the plant response to environmental stresses and ABA treatment. To elucidate the function of this gene,
recombinant plasmids expressing full-length ThPOD1 as well as ThPOD2 (aa 41-337), and ThPOD3 (aa 73-337) truncated polypeptides were constructed. SDS–PAGE and Western blot analyses of the fusion proteins revealed that
the molecular weights of ThPOD1, ThPOD2 and ThPOD3 were ~57, ~50 and ~47 kDa, respectively. Stress assays of E. coli treated with the recombinant plasmids indicated that ThPOD3 could improve resistance to drought stress. This finding could
potentially be used to improve plant tolerance to drought stress via gene transfer. 相似文献
4.
This study presents the binding of ovine factor H (fH) by various serotypes of Borrelia and simultaneously correlates their complement resistance to sheep serum. Affinity ligand binding assay was employed to study
the binding of borrelial proteins to ovine recombinant fH and its truncated forms (short consensus repeat, SCR 7 and SCRs
19–20). From a repertoire of 17 borrelial strains, only two strains showed affinity to sheep fH. A ~28-kDa protein of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s., strain SKT-2) bound full-length fH as well as SCRs 19–20. This fH-binding protein was further identified as complement
regulator-acquiring surface protein of B. burgdorferi (BbCRASP-1) by MALDI-TOF analysis. Surprisingly, a ~26-kDa protein of Borrelia bissettii (DN127) showed affinity to full-length fH but not to SCR 7 and SCRs19–20. In complement sensitivity assay, both strains—SKT-2
and DN127—were resistant to normal sheep serum. Significant complement resistance of two Borrelia garinii strains (G117 and T25) was also observed; however, none of those strains was able to bind sheep fH. Our study underscores
the need of further exploration of fH-mediated evasion of complement system by Borrelia in domestic animals. 相似文献
5.
V. D. Lazareva N. E. Bocharova K. E. Volynski T. M. Volkova E. V. Grishin 《Russian Journal of Bioorganic Chemistry》2000,26(4):271-276
α-Latrotoxin (α-LTX) binding sites to functionally active monoclonal antibodies (MA) A4 and A24 were localized using three
approaches: hydrolysis of the toxin followed by theN-terminal sequencing of immunoreactive peptides; the study of antibody interaction with several recombinant α-LTX fragments;
Western immunoblotting of synthetic overlapping peptides (6–8 aa) whose structures correspond to that of the immunoreactive
α-LTX fragment. It was shown that the MA A4 epitope is located within the F234-M294 protein fragment and that MA A24 interacts with the fragment347FDKDIT352. 相似文献
6.
Hui-Ping Chang Wan-Chi Liang Rui-Cin Lyu Meng-Chun Chi Tzu-Fan Wang Kuo-Liang Su Hui-Chih Hung Long-Liu Lin 《Biochemistry. Biokhimii?a》2010,75(7):919-929
The role of the C-terminal region of Bacillus licheniformis γ-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581–585, 577–585, 576–585, 566–585, 558–585, 523–585, and 479–585 amino acids, respectively, were generated by
site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing
of the enzyme, and the engineered protein was active towards the synthetic substrate L-γ-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Δ(576–585), pro-Δ(566–585), and pro-Δ(558–585), could partially precede a time-dependent autocatalytic
process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect
to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Δ(577–585), Δ(576–585), Δ(566–585), Δ(558–585), Δ(523–585),
and Δ(479–585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding
to half effect and ΔG
0 for the unfolding. BlGGT-4aa and BlGGT sedimented at ∼4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was
conserved in all of the truncated proteins. The frictional ratio (f/f
o) of BlGGT-4aa, BlGGT, Δ(581–585), and Δ(577–585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively
as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional
role of the C-terminal region in the autocatalytic processing of BlGGT. 相似文献
7.
Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 (map-1), slr0918 (map-2) and sll0555 (map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.Abbreviations map Gene encoding methionine aminopeptidase - MetAP Methionine aminopeptidase - eMetAP-Ia Escherichia coli methionine aminopeptidase type Ia - yMetAP-Ib Yeast methionine aminopeptidase type Ib - yMetAP-IIa Yeast methionine aminopeptidase type IIa - hMetAP-IIb Human methionine aminopeptidase type IIb - pfMetAP–IIa Pyrococcus furiosis methionine aminopeptidase type Ia - bst MetAP-Ia Bacillus stearothermophilus methionine aminopeptidase type Ia - c1MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-1 - c2MetAP-Ia Cyanobacterial methionine aminopeptidase type Ia encoded by map-2 - c3MetAP-Ib Cyanobacterial methionine aminopeptidase type Ib, ncoded by map-3 相似文献
8.
9.
The investigation of the recombinant bovine lactoferrin-derived antimicrobial protein (rBLfA) demonstrates that the inter-lobe
region of bovine lactoferrin contributes to iron binding stability and antimicrobial activity against Staphylococcus aureus. rBLfA containing N-lobe (amino acid residues 1–333) and inter-lobe region (residues 334–344) was expressed in Pichia pastoris at shaking flask and fermentor level. The recombinant intact bovine lactoferrin (rBLf) and N-lobe (rBLfN) were expressed
in the same system as control. The physical–chemical parameters of rBLfA, rBLfN and rBLf including amino acid residues, molecular
weight, isoelectric point, net positive charge and instability index were computed and compared. The simulated tertiary structure
and the calculated surface net charge showed that rBLfA maintained original structure and exhibited a higher cationic feature
than rBLf and rBLfN. The three proteins showed different iron binding stability and antimicrobial activity. rBLfA released
iron in the pH range of 7.0–3.5, whereas rBLfN lost its iron over the pH range of 7.0–4.0 and iron release from rBLf occurred
in the pH range of 5.5–3.0. However, the minimum inhibition concentration of rBLfA against S. aureus ATCC25923 was 6.5 μmol/L, compared with 12.5 and 25 μmol/L that of rBLfN and rBLf, respectively. These results revealed that
S. aureus was more sensitive to rBLfA than rBLfN and rBLf. It appeared that the strong cationic character of inter-lobe region related
positively to the higher anti-S. aureus activity. 相似文献
10.
Ali Dehestani Kamal Kazemitabar Gholamreza Ahmadian Nadali Babaeian Jelodar Ali Hatef Salmanian Mehdi Seyedi Heshmat Rahimian Seyedhadi Ghasemi 《Biotechnology letters》2010,32(4):539-546
The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed
that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant
difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore
germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains
are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of
fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity
causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system. 相似文献
11.
Likhareva Viktoria V. Mikhailova Anna G. Vaskovsky Boris V. Garanin Sergey K. Rumsh Lev D. 《International journal of peptide research and therapeutics》2002,9(2-3):71-76
Summary Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency
hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for
the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive
peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF)
(AT). Model peptides with truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic
parameters of enteropeptidase hydrolysis for these substrates were determined.K
m values for all substrates with truncated linker (≈10−3 M) are an order of magnitude higher than corresponding values for typical enteropeptidase artificial peptide or fusion protein
substrates with full enteropeptidase linker-DDDDK-(K
m
≈10−4 M).k
cat values for AT, Hb (2–8), WDDRG and WDDKG are ≈30–40 min−1. But one additional amino acid residue at both N-and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency:k
cat value for Hb (1–9) is 1510 min−1. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned
truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active
peptidesin vitro along with its unique natural substrate trypsinogen was demonstrated. 相似文献
12.
Kim YM Shimizu R Nakai H Mori H Okuyama M Kang MS Fujimoto Z Funane K Kim D Kimura A 《Applied microbiology and biotechnology》2011,91(2):329-339
Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66
Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90
was proteolytically degraded to more than seven polypeptides (23–70 kDa) during long storage. The protease-insensitive protein
was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from
the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable
region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid
of C-VR (TM-NCGΔ) or N-VR/C-VR (TM-ΔCGΔ) were catalytically active, thereby indicating that N-VR and C-VR were not essential
for the catalytic activity. TM-ΔCGΔ did not accept any further protease-degradation during long storage. TM-NCGΔ and TM-ΔCGΔ
enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site. 相似文献
13.
Wang XF Dong CF Zhang J Wan YZ Li F Huang YX Han L Shan B Gao C Han J Dong XP 《Molecular and cellular biochemistry》2008,310(1-2):49-55
Microtubule associated protein tau is considered to play roles in some types of human transmissible spongiform encephalopathies
(TSE). In this study, the full-length and several truncated human tau proteins were expressed from E. coli and purified. Using GST pull down, co-immunoprecipitation assay and tau-coated ELISA, the molecular interaction between tau
protein and PrP was confirmed in the context of the full-length human tau. The N terminus (amino acids 1–91) and tandem repeats
region (amino acids 186–283) of tau protein were responsible for the interaction with PrP. The octapeptide repeats within
PrP directly affected the binding activity of PrP with tau. GSS-related mutant PrP102L and fCJD- related mutants with two
and seven extra octarepeats showed more active binding capacity with tau than wild-type PrP. The molecular interactions between
PrP and tau protein highlight a potential role of tau in the biological function of PrP and the pathogenesis of TSE. 相似文献
14.
Recombinant human progastrin6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [Baldwin (2004) Protein J 23:65–70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not
they bound ferric ions. Recombinant rat and human procholecystokinin57–95 were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant
procholecystokinin57–95 did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition
of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated
that binding to procholecystokinin57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin6–80. 相似文献
15.
构建汉滩病毒76—118N蛋白及其分别从N-端和C-端缺失的共6个突变体,在大肠杆菌BL-21中进行表达,并对其中一些蛋白进行了纯化。通过Western blot、酶联免疫吸附试验(ELISA)进行汉滩病毒N蛋白的抗原表位分析,N蛋白及6个缺失突变体都与组特异性抗体L13F3呈阳性反应,而缺失突变体与型特异性抗体AH30呈阴性反应。构建汉滩病毒76—118N蛋白及其6个缺失突变体的真核表达载体,并在COS-7细胞中进行表达。通过间接免疫荧光试验(IFA)进行汉滩病毒N蛋白的抗原表位分析,病人血清与真核表达的N蛋白及6个缺失突变体呈阳性反应。而仅有N蛋白及缺失N端1~30位氨基酸序列的NPN30与型特异性抗体AH30呈阳性反应。证实组特异性抗体L13F3结合的抗原表位位于N端1~30位氨基酸;而C端抗原表位对于型特异性抗体AH30与N蛋白的识别和结合具有重要意义,缺失N端100位氨基酸序列可能破坏羧基端构象型表位,也可以影响N蛋白与AH30的结合。 相似文献
16.
Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2
proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution
in the form of complexes of 2–4 protein molecules of 12–18 nm size. We used computer methods to design models of complexes
consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with
ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering.
We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial
length of the ssDNA. 相似文献
17.
Lee HK Lee BH Dutta NK Seok SH Baek MW Lee HY Kim DJ Na YR Noh KJ Park SH Kariwa H Nakauchi M Mai le Q Heo SJ Park JH 《Journal of microbiology and biotechnology》2008,18(10):1717-1721
Severe acute respiratory syndrome (SARS) is a lifethreatening emerging respiratory disease caused by the coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is highly antigenic and may be a suitable candidate for diagnostic applications. We constructed truncated recombinant N proteins (N1 [1-422 aa], N2 [1- 109 aa], and N3 [110-422 aa]) and determined their antigenicity by Western blotting using convalescent SARS serum. The recombinants containing N1 and N3 reacted with convalescent SARS serum in Western blotting. However, the recombinant with N2 did not. In ELISA using N1 or N3 as the antigens, positive results were observed in 10 of 10 (100%) SARS-CoV-positive human sera. None of 50 healthy sera gave positive results in either assay. These data indicate that the ELISA using N1 or N3 has high sensitivity and specificity. These results suggest that the middle or C-terminal region of the SARS N protein is important for eliciting antibodies against SARS-CoV during the immune response, and ELISA reactions using N1 or N3 may be a valuable tool for SARS diagnosis. 相似文献
18.
The pentatricopeptide repeat (PPR) protein family is one of the largest and most complex families in plants. These proteins
contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding RNA. PPR proteins have been
implicated in many crucial functions broadly involving organelle biogenesis and plant development. In this study, we identified
many genes encoding PPR protein in Upland cotton through an extensive survey of the database of Gossypium hirsutum. Furthermore, we isolated five full-length cDNA of PPR genes from G. hirsutum 0-613-2R which were named GhPPR1–GhPPR5. Domain analysis revealed that the deduced amino acid sequences of GhPPR1–5 contained
from 5 to 10 PPR motifs and those PPR proteins were divided into two different PPR subfamilies. GhPPR1–2 belonged to the PLS
subfamily and GhPPR3–5 belonged to the P subfamily. Phylogenetic analysis of the five GhPPR proteins and 18 other plant PPR
proteins also revealed that the same subfamily clustered together. All five GhPPR genes were differentially but constitutively
expressed in roots, stems, leaves, pollens, and fibers based on the gene expression analysis by real-time quantitative RT-PCR.
This study is the first report and analysis of genes encoding PPR proteins in cotton. 相似文献
19.
Hajnalka Szabados Szilvia Bősze Pálma Silló Sarolta Kárpáti Ferenc Hudecz Katalin Uray 《Journal of peptide science》2013,19(2):84-94
Desmosomal transmembrane glycoproteins desmogleins (Dsg) 1 and 3 are targets of life‐threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus (PF). In these diseases, pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell–cell adhesion and clinically, to the presence of blisters and erosions. Identification, characterization, and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding the immunopathology of the disease and also in the design of novel diagnostics. Here, we describe the localization of the B‐cell epitope regions of Dsg1 and Dsg3 proteins' extracellular parts recognized by IgG‐type serum autoantibodies of patients with PV and PF. In our study, overlapping pentadecapeptides were synthesized on hydroxypropyl methacrylate pins based on the results of in silico predictions. To detect the interaction between the serum autoantibodies and the immobilized synthetic peptides, modified Enzyme Linked Immunosorbent Assay (ELISA) was performed with pin‐attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86‐110, aa196‐220, aa226‐250, aa326‐340, and aa486‐520) within the extracellular part of the Dsg1 and four possible epitope regions (aa64‐78, aa330‐344, aa375‐399, and aa446‐460) within that of the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin‐bound overlapping peptides in modified ELISAs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
20.
以戊型肝炎病毒(HEV)第4基因型中国株ORF2编码蛋白p166Chn制备单克隆抗体(McAbs), 同时制备20个N端或C端逐渐截短的p166Chn截短蛋白, 与7种不同基因型和亚型的p166蛋白一起, 通过ELISA、免疫印迹(Western blot)以及竞争抑制实验对主要存在于我国的HEV第4基因型毒株进行抗原表位分析。结果发现所制备的McAbs与p166Chn截短蛋白的免疫反应有两种类型, 以1G10为代表的McAbs能与N端不短于aa477、C端不短于aa613的截短蛋白反应, 其针对的抗原表位是构象依赖型表位, 依赖于aa477~aa613肽链区段; 而McAb 2F11则能与N端不短于aa474、C端不短于aa617的截短蛋白反应, 其针对的抗原表位也是构象表位, 但需依赖于较长的肽链区段(aa474~aa617)。竞争抑制实验显示两类McAbs互不抑制, 进一步证实了所发现的两个抗原表位在空间位置上的不同。更有意义的是, 两类McAbs均能与其他不同HEV基因型和亚型来源的p166重组蛋白发生阳性反应, 表明这两个抗原表位是HEV基因型共同性的, 可以在世界各国分布的不同基因型HEV毒株中诱导交叉免疫。 相似文献