共查询到20条相似文献,搜索用时 15 毫秒
1.
Jong Won Han Kang Sup Yoon Tatyana A. Klochkova Mi-Sook Hwang Gwang Hoon Kim 《Journal of applied phycology》2011,23(4):745-753
A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and
MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein
with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal
and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed
no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from
PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the
cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously
reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence
of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails. 相似文献
2.
Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris 总被引:1,自引:0,他引:1
cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional. 相似文献
3.
Yanyang Liu Junzhou Li Yuling Li Mengguan Wei Qingxin Cui Qilei Wang 《Molecular biology reports》2010,37(2):755-761
A full-length cDNA encoding a maize GTP-binding protein of the ADP-ribosylation factor family was cloned by suppression subtractive
hybridization and an in silico cloning approach. The cDNA was 938 bp in length and contained a complete ORF of 612 bp, which
encodes a protein of 203 amino acid residues. Its deduced amino acids sequence had an 83% identity with that of a GTP-binding
protein in rice. The gene was designated ZmArf2. The ZmArf2 gene consists of G1, G2, G3, G4 and G5 boxes, and Switch I and Switch II regions. Eight nucleotides differed and five amino
acids changed between the popcorn inbred N04 and the dent corn inbred Dan232. One changed amino acid was in the G1 box. RT-PCR
analysis showed that ZmArf2 expression increased in the early stages of endosperm development and was not tissue-specific. 相似文献
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Jinlin Zhou Min Liao Haiyan Gong Xuenan Xuan Kozo Fujisaki 《Experimental & applied acarology》2010,51(4):327-333
The gene encoding cystatin from the tick Haemaphysalis longicornis has been reported previously. In the study reported here, we characterized a member of cystatins and designated it as Hlcyst-3
(H. longicornis cystatin-3). Its full-length cDNA is 602 bp, and it encodes a putative 129 amino acid protein with an obvious signal peptide. Sequence
analysis revealed that it has significant homology with the known secreted cystatin. The recombinant protein was expressed
in a GST-fused soluble form in Escherichia coli and was purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain and cathepsin
L was identified by fluorogenic substrate analysis. Real-time PCR revealed that Hlcyst-3 was mostly expressed in the tick
midgut. 相似文献
6.
A chalcone reductase (CHR) gene was isolated from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). The full-length cDNA of A. mongholicus CHR, designated as Amchr (GenBank accession No. HM357239), was 1196 bp long. It had a 957 bp open reading frame encoding a 318-amino acid protein
of 35 kDa, a 67 bp 5′ non-coding region and a 172 bp 3′-untranslated region. The putative AmCHR protein showed striking similarity
to CHR from other leguminous species. Two-dimensional structure modeling showed that AmCHR consisted of 45.28% α-helix, 10.38%
extended strand and 44.34% random coil. Prediction showed that three-dimensional AmCHR was a global protein containing an
aldo-ket-red domain, with a putative Asp-Tyr-Lys-His catalytic tetrad in the center. The AmCHR gene was 1251 bp long, consisting
of three exons and two introns. Intron I was 125 bp and intron II was 169 bp long. Southern blot analysis indicated that Amchr belonged to a small multigene family. Under natural conditions, Amchr was expressed differentially in the root, stem and leaf tissues of A. mongholicus, with a preferential expression in the root. The recombinant AmCHR protein was successfully expressed in Escherichia coli strain BL21 with pET42a vector. The result showed that the expressed AmCHR protein had molecular weight of about 35 kDa,
which matched the size of the predicted protein by bioinformatic analysis. This study opened avenues towards understanding
of the function of AmCHR protein and the role of the Amchr gene in the calycosin-7-O-β-d-glucoside branch pathway in A. mongholicus. 相似文献
7.
Y. Li S. L. Yang Z. L. Tang W. T. Cui Y. L. Mu M. X. Chu S. H. Zhao Z. F. Wu K. M. Peng K. Li 《Molecular biology reports》2010,37(3):1309-1317
As a component of E3 ubiquitin protein ligases called SCFs, SKP2 protein belongs to a member of FBLs protein which is the biggest eukaryotic subfamily of F-BOX proteins with 12 members.
In this study, we cloned and sequenced partial cDNA, intron 1 and intron 6 of porcine SKP2 gene. The partial cDNA is 1,402 bp long and has an open reading frame of 1,272 bp which encodes 424 putative amino acids.
The deduced protein comprises a conserved F-BOX domain at position from the 90th to 140th amino acid. The phylogenetic tree
indicated that porcine SKP2 has the closest genetic relationship with bovine SKP2 than other selected animal species. Quantitative RT-PCR analysis displayed that the tissue expression level of porcine SKP2 fluctuated remarkably in a large range, and it expressed in thymus with the highest level and in longissimus dorsi muscle
with the lowest level. Two SNPs were identified, meanwhile, further polymorphism analysis with Cfr42I showed that AA genotype
was in dominance absolutely among four kinds of unrelated Chinese indigenous miniature and one introduced Landrace pig breeds.
In addition, association analysis with immune traits and blood parameters revealed that the SNP Cfr42I in intron 1 was significantly
associated with red cell distribution width of neonate piglets at 0 day (P = 0.027). 相似文献
8.
In-Hye Park Jie Chang Yong-Seok Lee Shu-Jun Fang Yong-Lark Choi 《The protein journal》2012,31(3):238-245
The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal
peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase
was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight
of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C
and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained
66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl
β-d-cellobiose, but no activity was detected against p-nitrophenyl β-d-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography,
while enzyme did not hydrolyze cellobiose and cellotriose. 相似文献
9.
A novel phenylacetic acid (PAA)-induced CoA-ligase-encoding gene, designated as phlC, has been cloned from penicillin-producing fungus Penicillium chrysogenum. The open reading frame of phlC cDNA was 1671 bp and encoded a 556 amino acid residues protein with the consensus AMP binding site and a peroxisomal targeting
signal 1 on its C terminus. The deduced amino acid sequence showed 37% and 38% identity with characterized P. chrysogenum Phl and PhlB protein, respectively. Functional recombinant PhlC protein was overexpressed in Escherichia coli. The purified recombinant enzyme was capable to convert PAA into its corresponding CoA ester with a specific activity of
129.5 ± 3.026 pmol/min per mg protein. Similar to Phl and PhlB, PhlC displayed broad substrate spectrum and showed higher
activities to medium- and long-chain fatty acids. The catalytic properties of PhlC have been determined and compared to those
of Phl and PhlB. 相似文献
10.
Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative
analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative
RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein
was successfully expressed in Escherichia coli with the molecular weight expected. 相似文献
11.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
12.
EJO1, a novel gene presumably involved in the ovary development of the Chinese mitten crab (Eriocheir japonica sinensis), was identified and characterized by suppression subtractive hybridization and cDNA macroarray analysis. EJO1 expression was 2.6-fold higher at stage III than at stage II during the ovary development of the mitten crab. EJO1 is 876 bp in length containing a 759 bp open reading frame which encodes a 252-amino-acid protein. Homology analysis showed
that no sequence significantly matching EJO1 was found in SwissProt, so it was deduced as a novel gene (GenBank accession number: AY185917). The EJO1 protein is probably
a secretion protein with a signal peptide of 17 amino acids. The pI/Mw deduced from the amino acid sequence was 6.18/28.18 kDa.
Expression profile showed that EJO1 mRNA is highly expressed in the heart, intestine, and ovary of the crab, while there is little or no expression in muscle
and hepatopancreas. The differential expression of EJO1 at the different developmental stages of the ovary was further confirmed by Northern blot analysis. In conclusion, EJO1 is a novel gene differentially expressed in the ovary of the Chinese mitten crab, which may play an important role in the
ovary development. 相似文献
13.
Cloning and sequence analysis of a novel xylanase gene, Auxyn10A, from Aspergillus usamii 总被引:1,自引:0,他引:1
A full-length cDNA sequence, encoding a novel endo-1,4-β-d-xylanase (AuXyn10A) of Aspergillus usamii, was obtained by using rapid amplification of cDNA ends (RACE) methods and cloned into the pUCm-T vector, followed by DNA
sequencing. The cDNA gene, designated as Auxyn10A, is 1,235 bp in length harboring 5′- and 3′-non-encoding regions, as well as an ORF of 984 bp that encodes a 19-aa signal
peptide, a 6-aa propeptide and a 302-aa mature peptide with a calculated MW of 32,756 Da. The AuXyn10A displays high similarity
to the xylanases of Aspergillus niger, Aspergillus kawachii and Aspergillus niger, members of the glycoside hydrolase family 10. Its three-dimensional structure was predicted using programs based on the crystal structure of Penicillium simplicissimum xylanase (1B30_A) from the family 10. The complete DNA gene was cloned from the genomic DNA of A. usamii using conventional PCR and hairpin structure-mediated PCR techniques. The DNA gene is 2,255 bp in length, containing a 510 bp
of 5′-flanking promoter region and a 1,745 bp of downstream fragment that consists of ten exons and nine short introns ranging
from 52 to 62 bp. 相似文献
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Bin Liu Wenguo Wang Jihai Gao Fang Chen Shenghua Wang Ying Xu Lin Tang Yongjiong Jia 《Acta Physiologiae Plantarum》2010,32(3):531-539
Herein, we cloned a full-length cDNA encoding allene oxide cyclase (AOC, EC 5.3.99.6) that is a key enzyme in jasmonates (JAs)
biosynthetic pathway from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels, named as JcAOC (GenBank accession no. FJ874630). The cDNA was 924 bp in length with a complete open reading frame of 750 bp, which encoded
a polypeptide of 250 amino acids including a putative signal peptide of 65 amino acid residues and a mature protein of 185
amino acids with a predicted molecular mass of 20.7 kDa and a isoelectric point of 6.24. Phylogenetic analysis indicated that
JcAOC belonged to the AOC superfamily. Semi-quantitative RT-PCR analysis revealed that JcAOC mRNA was expressed in roots, stems, leaves, young seeds, endosperms, and flowers, but that the expression level was highest
in leaves and lowest in seeds, and mRNA expression of JcAOC could be induced by salt stress (300 mM NaCl) and low temperature (4°C). Furthermore, the full-length coding region of JcAOC excluding signal peptide sequence was inserted into pET-30a and was successfully expressed in Escherichia coli. Overexpression of JcAOC in E. coli conferred its resistance to salt stress and low temperature. 相似文献
20.
《Bioscience, biotechnology, and biochemistry》2013,77(9):2256-2265
cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5′ untranslated region (UTR) of 869 bp, and the 3′ UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase. 相似文献