共查询到20条相似文献,搜索用时 24 毫秒
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Esper Boel Birgitte Huge-Jensen Mogens Christensen Lars Thim Niels P. Fiil 《Lipids》1988,23(7):701-706
ARhizomucor miehie cDNA library constructed inEscherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride
lipase (triacyl-glycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their
insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a
precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269
residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine
residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified
in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron
in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesizedR. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing. 相似文献
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Maoka T Tanimoto F Sano M Tsurukawa K Tsuno T Tsujiwaki S Ishimaru K Takii K 《Journal of oleo science》2008,57(2):133-137
The effects of ferulic acid (FA) and gamma-oryzanol (OZ) supplementation on cultured red sea bream were examined. Commercial brown fish meal diets supplemented with FA (0.01-0.5%) or OZ (0.05-0.5%) were given to zero-year, cultured red sea bream for 98 days. After the experiment, the brightness of the integument color ("L" value) of FA- and OZ-administrated fish was higher than that of control fish. Furthermore, 2-Thiobarbituric acid reactive substances (TBARS) in the liver of FA- and OZ-administrated fish was lower than in control fish. These results indicate that FA and OZ suppressed not only dark-color pigmentation but also oxidative stress in cultured red sea bream. 相似文献
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A cDNA encoding protein with homology to plant secretory phospholipase A2 (sPLA2), denoted as Nt1 PLA2, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature
form of Nt1 PLA2 has 12 cysteines, Ca2+ binding loop and catalytic site domain that are commonly conserved in plant sPLA2s. The recombinant Nt1 PLA2 was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease
to obtain the mature form. Interestingly, Nt1 PLA2 could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for
the sn-1 and -2 positions of glycerophospholipids required Ca2+ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine
was used as a substrate. The level of Nt1 PLA2 mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid. 相似文献
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目的 以鸡脾细胞mRNA为模板扩增编码鸡IL 18成熟蛋白的cDNA ,并在大肠杆菌中进行表达。方法 用PHA和LPS激活的AA肉鸡脾细胞 ,提取mRNA ,以RT PCR法扩增出编码鸡IL 18成熟蛋白的cDNA并测序 ,与pET 2 8b载体构建重组质粒 ,并在大肠杆菌BL2 1(DE3)plysS中表达 ,用SDS PAGE检测表达产物。结果 所克隆的核苷酸片段包含了鸡IL 18全部成熟蛋白编码基因 ,其大小为 5 0 7个核苷酸 ,编码 16 9个氨基酸 ;构建的重组表达载体在大肠杆菌BL2 1(DE3)plysS中表达出相对分子质量为 190 0 0的重组蛋白。结论 已成功克隆和表达了鸡IL 18成熟蛋白基因。 相似文献
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Phospholipase A2 (PLA2) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA2 activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA2 activity in the flower appears to have originated from both Ca2+-dependent and-independent PLA2. A cDNA clone for protein with homology to animal secretory PLA2 (sPLA2), denoted as Nt PLA2, was isolated from the tobacco flower. The cDNA of Nt PLA2 encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence
for mature Nt PLA2 contains 12 cysteines, a Ca2+ binding loop, and a catalytic domain that are commonly conserved in animal sPLA2. The Nt PLA2 mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA2 was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then
the recombinant mature Nt PLA2 was purified by ion exchange chromatography. It was discovered that the purified Nt PLA2 essentially requires Ca2+, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate.
The optimal activity of Nt PLA2 was at pH 8–10 when PC was used as a substrate. 相似文献
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Wen-Kai Xia Tian-Bo Ding Jin-Zhi Niu Chong-Yu Liao Rui Zhong Wen-Jia Yang Bin Liu Wei Dou Jin-Jun Wang 《International journal of molecular sciences》2014,15(3):3711-3728
Chitin synthase synthesizes chitin, which is critical for the arthropod exoskeleton. In this study, we cloned the cDNA sequences of a chitin synthase 1 gene, PcCHS1, in the citrus red mite, Panonychus citri (McGregor), which is one of the most economically important pests of citrus worldwide. The full-length cDNA of PcCHS1 contains an open reading frame of 4605 bp of nucleotides, which encodes a protein of 1535 amino acid residues with a predicted molecular mass of 175.0 kDa. A phylogenetic analysis showed that PcCHS1 was most closely related to CHS1 from Tetranychus urticae. During P. citri development, PcCHS1 was constantly expressed in all stages but highly expressed in the egg stage (114.8-fold higher than in the adult). When larvae were exposed to diflubenzuron (DFB) for 6 h, the mite had a significantly high mortality rate, and the mRNA expression levels of PcCHS1 were significantly enhanced. These results indicate a promising use of DFB to control P. citri, by possibly acting as an inhibitor in chitin synthesis as indicated by the up-regulation of PcCHS1 after exposure to DFB. 相似文献
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目的克隆TNF家族B细胞激活因子(BcellactivatingfactortotheTNFfamily,BAFF)胞外区134~285(sBAFF134-285)氨基酸残基段缺失突变体(sΔBAFF)的cDNA,并进行表达及纯化。方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向PCR法,扩增缺失编码sBAFF的142~160位氨基酸的核苷酸序列。经测序证实后,克隆入原核表达载体pQE-80L。经IPTG诱导表达,SDS-PAGE和Westernblot检测表达产物,Ni2+-NTA柱层析纯化目的蛋白。结果经一步反向PCR扩增后得到401bp的DNA片段,该片段序列与GenBank报道的编码人ΔBAFF的胞外区(sΔBAFF)cDNA序列一致。含sΔBAFF的表达载体在大肠杆菌中可表达出相对分子质量为18000的蛋白质并以包涵体的形式存在,经Ni2+-NTA柱层析纯化后得到高纯度的目的蛋白。结论已成功地制备出人sΔBAFF蛋白,为其功能的研究创造了条件。 相似文献
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目的分析赤子爱胜蚓蛋白磷酸酶2A(protein phosphatase 2A,PP2A)类似激活剂样蛋白的cDNA和氨基酸序列。方法从赤子爱胜蚓cDNA文库中随机测序得到目标cDNA序列,应用DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL、PDB等基因和蛋白质分析软件进行该目标基因测序及氨基酸序列分析。结果赤子爱胜蚓PP2A类似激活剂样蛋白cDNA序列长547 bp,编码87个氨基酸残基;与该蛋白基因序列相似度较高的多是不同物种中PP2A类似激活剂蛋白基因序列,其中与Crassostrea virginica serine/threonine-protein phosphatase 2A activator-like mRNA核苷酸相似度为67%;与该蛋白氨基酸序列相似度较高的多是不同物种中的PP2A类似激活剂蛋白氨基酸序列,其中与serine/threonine-protein phosphatase 2A activator(Crassostrea gigas)氨基酸相似度为70%;目标序列中存在一段PTPA超家族保守结构域,蛋白质二级结构以α螺旋以及无规则卷曲为主,三级预测结果与二级结构一致。结论本实验分析的赤子爱胜蚓PP2A类似激活剂样蛋白与牡蛎PP2A类似激活剂蛋白及存在于所有真核生物中且高度保守的PTPA同源度较高,可能具有特异性激活肿瘤抑制因子PP2A的间接抑制肿瘤作用。 相似文献
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Ward RJ; Alves AR; Ruggiero Neto J; Arni RK; Casari G 《Protein engineering, design & selection : PEDS》1998,11(4):285-294
'SequenceSpace' analysis is a novel approach which has been used to
identify unique amino acids within a sub-family of phospholipases A2 (PLA2)
in which the highly conserved active site residue Asp49 is substituted by
Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor
Ca2+ and possess extremely low catalytic activity, they demonstrate a
Ca2+-independent membrane damaging activity through a poorly understood
mechanism, which does not involve lipid hydrolysis. Additionally,
Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic
pharmacological activities, however the structural basis of these varied
functions is largely unknown. Using the 'SequenceSpace' analysis we have
identified nine residues highly unique to the Lys49- PLA2 sub-family, which
are grouped in three amino acid clusters in the active site, hydrophobic
substrate binding channel and homodimer interface regions. These three
highly specific residue clusters may have relevance for the
Ca2+-independent membrane damaging activity. Of a further 15 less
stringently conserved residues, nine are located in two additional clusters
which are well isolated from the active site region. The less strictly
conserved clusters have been used in predictive sequence searches to
correlate amino acid patterns in other venom PLA2s with their
pharmacological activities, and motifs for presynaptic and combined
toxicities are proposed.
相似文献
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目的克隆新的小鼠激活素受体相互作用蛋白5(ActRIP5)基因。方法应用酵母双杂交技术,筛选出与激活素ⅡA型受体(ActRⅡA)相互作用蛋白的基因片段,再以此基因片段作为探针,从小鼠脑cDNA文库中钓取ActRIP5cDNA。采用哺乳动物细胞双杂交系统,进一步确定ActRIP5与ActRⅡA的相互作用。采用RT-PCR检测ActRIP5mRNA在组织中的转录。结果克隆的ActRIP5全编码基因长1839bp,编码145个氨基酸残基,与ActRⅡA具有特异结合作用。ActRIP5mR-NA在小鼠多种组织中均可检出。结论ActRIP5属于ActRIP家族新成员,可以与ActRⅡA相互作用。 相似文献
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Nan-Nan Liu Zeng-Shan Liu Pan Hu Ying Zhang Shi-Ying Lu Yan-Song Li Yong-Jie Yang Dong-Song Zhang Yu Zhou Hong-Lin Ren 《International journal of molecular sciences》2016,17(8)
Lysophospholipase I (LYPLA1) is an important protein with multiple functions. In this study, the full-length cDNA of the LYPLA1 gene from Ovis aries (OaLypla1) was cloned using primers and rapid amplification of cDNA ends (RACE) technology. The full-length OaLypla1 was 2457 bp with a 5′-untranslated region (UTR) of 24 bp, a 3′-UTR of 1740 bp with a poly (A) tail, and an open reading frame (ORF) of 693 bp encoding a protein of 230 amino acid residues with a predicted molecular weight of 24,625.78 Da. Phylogenetic analysis showed that the OaLypla1 protein shared a high amino acid identity with LYPLA1 of Bos taurus. The recombinant OaLypla1 protein was expressed and purified, and its phospholipase activity was identified. Monoclonal antibodies (mAb) against OaLypla1 that bound native OaLypla1 were generated. Real-time PCR analysis revealed that OaLypla1 was constitutively expressed in the liver, spleen, lung, kidney, and white blood cells of sheep, with the highest level in the kidney. Additionally, the mRNA levels of OaLypla1 in the buffy coats of sheep challenged with virulent or avirulent Brucella strains were down-regulated compared to untreated sheep. The results suggest that OaLypla1 may have an important physiological role in the host response to bacteria. The function of OaLypla1 in the host response to bacterial infection requires further study in the future. 相似文献
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《中国生物制品学杂志》2010,(12)
目的克隆并分析籽鹅卵巢产蛋性能相关基因EST1的全长cDNA序列。方法利用实时荧光定量PCR技术对EST1基因在籽鹅产蛋前期与产蛋期卵巢中mRNA表达水平进行检测,并采用RACE(Rapid amplification of cDNAends,RACE)技术对该基因全长cDNA序列进行克隆,应用生物信息学预测方法对其编码的蛋白质进行分析。结果籽鹅产蛋期卵巢组织中EST1基因mRNA的表达水平显著高于产蛋前期(P<0.05)。经RACE技术获得EST1基因全长cDNA序列长1715bp,具有单一的完整开放阅读框(ORF,14~1318bp),推测编码蛋白含434个氨基酸残基,相对分子质量为107100,等电点为5.00。该蛋白为细胞质内蛋白,含3个跨膜螺旋,蛋白序列中含1个信号肽切割位点。结论经分子生物学软件进行蛋白质功能预测,初步确定EST1基因为籽鹅α-烯醇化酶蛋白基因,推测该基因可能参与籽鹅产蛋性能的分子调控。 相似文献
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The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides
in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The
open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide,
with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology
with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis
and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were
not changed when compared with its natural counterpart.
Zhikang Qian and Peihong Jiang are equal contributors as first author of this paper. 相似文献
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Yu Jiao Zhe Zheng Rongrong Tian Xiaodong Du Qingheng Wang Ronglian Huang 《International journal of molecular sciences》2015,16(9):21442-21453
MicroRNAs (miRNAs) are noncoding RNA molecules that function as negative regulators of target genes. In our previous research, 258 pm-miRNAs were identified in Pinctada martensii by Solexa deep sequencing. Pm-miR-2305 was one of the identified pm-miRNAs with a potential function in biomineralization. In the present study, the precursor of pm-miR-2305 was predicted with 96 bp, containing a characteristic hairpin structure. Stem-loop qRT-PCR analysis indicated that pm-miR-2305 was constitutively expressed in all the tissues (adductor muscle, gill, mantle, hepatopancreas, foot, and gonad) of P. martensii and was highly expressed in the foot. After the over-expression of pm-miR-2305 in the mantle by mimics injection into the muscle of P. martensii, nacre demonstrated disorderly growth, as detected by scanning electron microscopy. Dual luciferase reporter gene assay indicated that pm-miR-2305 mimics could significantly inhibit the luciferase activity of the reporter containing the 3′UTR of the pearlin gene. Western blot analysis demonstrated that the protein expression of pearlin was down-regulated in the mantle tissue after the over-expression of pm-miR-2305. Therefore, our data showed that pm-miR-2305 participated in nacre formation by targeting pearlin in P. martensii. 相似文献
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Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions 总被引:1,自引:0,他引:1
Through three cumulative amino acid substitutions, we constructed novel
mutant subtilisins E of Bacillus subtilis, all with high specificity,
activity and productivity. The substitution of conserved Gly127,
constituting P1 substrate-binding pocket, with Ala and Val showed a marked
preference for the small P1 substrate. Leu was then substituted for Ile31
next to the catalytic Asp32 to enhance the catalytic activity. Both double
mutants (I31L/G127A and I31L/G127V) showed a 3-5- fold increase in
catalytic efficiency due to a large kcat, without any change in the
specificity of the mutants at position 127. Molecular modeling suggests
that large P1 residues were unable to access the pocket because of steric
hindrance. A third mutation was introduced by replacing Tyr(-1) with Ala in
the propeptide essential for autoprocessing to active mature subtilisin in
vivo. A prominent 7-20- fold increase in active enzyme production occurred
in the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).
相似文献
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Sequence properties of GPI-anchored proteins near the omega-site: constraints for the polypeptide binding site of the putative transamidase 总被引:8,自引:0,他引:8
Eisenhaber B; Bork P; Eisenhaber F 《Protein engineering, design & selection : PEDS》1998,11(12):1155-1161
Glycosylphosphatidylinositol (GPI) anchoring is a common post-
translational modification of extracellular eukaryotic proteins. Attachment
of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide
occurs after proteolytic cleavage of a C-terminal propeptide. In this work,
the sequence pattern for GPI-modification was analyzed in terms of physical
amino acid properties based on a database analysis of annotated proprotein
sequences. In addition to a refinement of previously described sequence
signals, we report conserved sequence properties in the regions omega -
11...omega - 1 and omega + 4...omega + 5. We present statistical evidence
for volume-compensating residue exchanges with respect to the positions
omega - 1...omega + 2. Differences between protozoan and metazoan
GPI-modification motifs consist mainly in variations of preferences to
amino acid types at the positions near the omega-site and in the overall
motif length. The variations of polypeptide substrates are exploited to
suggest a model of the polypeptide binding site of the putative
transamidase, the enzyme catalyzing the GPI-modification. The volume of the
active site cleft accommodating the four residues omega - 1...omega + 2
appears to be approximately 540 A3.
相似文献