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1.
The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme. 相似文献
2.
Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum 总被引:1,自引:0,他引:1
Sabine Merkelbach Johanna Gehlen Martin Denecke Heinz-Josef Hirsch Fritz Kreuzaler 《Plant molecular biology》1993,23(4):881-888
A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC-
Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes. 相似文献
3.
Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
4.
Using a fluorescent whole-cell Escherichia coli biosensor previously developed in our laboratory, we determined total and bioavailable lysine in four feed ingredients (soybean,
cottonseed, meat and bone meal, and sorghum) and three complete feeds (chick starter and finisher, and swine starter). The
same feed sources were analyzed for total lysine by high performance liquid chromatography (HPLC) and bioavailable lysine
by chick bioassay. No significant differences were found between bioavailable lysine estimates for soybean, cottonseed, meat
and bone meal, chick starter and finisher, and swine starter obtained by the fluorescent E. coli biosensor and chick bioassay. Except for sorghum, the E. coli biosensor estimates for total lysine were highly comparable to those obtained by HPLC. Comparisons were also conducted between
conventionally performed optical density-based and the newly developed fluorescence-based lysine assay. The lack of significant
differences in data obtained for total and bioavailable lysine by both detection modes indicated reliance and accuracy of
the fluorescent E. coli biosensor. Overall results suggest that the microbial assay based on green fluorescent protein fluorescence represents a
promising alternative method for lysine quantification. 相似文献
5.
A cDNA coding mutated cecropin CMIV fromBombyx mori was synthesized according to its amino acid sequence usingE. coli biased codons. The gene was cloned into the fusion expression vector pEZZ318 and was expressed inE. coli HB101. The fusion protein produced was purified by affinity chromatography to yield 26 mg/L fusion product. The anti-bacterial
activities of recombinant cecropin CMIV were recovered after cleavage by chemical method. 相似文献
6.
Two cDNA fragments (lrF1 and lrF2) representing a fibrinolytic enzyme gene of F-III-2 (GenBank AB045719), without and with
signal peptide coding sequence, were cloned from earthworm Lumbricus rubellus. The two fragments were inserted into bacterial expression vector pET28a (+), respectively. Subsequent expression showed
that both lrF1 and lrF2 proteins were produced as an inclusion body form in E. coli BL21 (DE3) pLysE. After protein refolding and purification, the fusion lrF1 and its derivative without poly histidine tags
at the N-terminus showed fibrinolytic activity on fibrin plates with relative activity of 134.3 U/mg protein and 139.7 U/mg
protein, respectively, whereas the fusion lrF2 and its derivative without the tags at the N-terminus, had no fibrinolytic
activity. The results indicated that the E. coli expression system could not recognize the endogenous signal peptide of F-III-2, and the effect of the histidine tags at the
N-terminus on the fibrinolytic activity of the expressed protein was insignificant. 相似文献
7.
Escherichia coli PrlC is a trypsin-like proteinase regulating the cell cycle. The Escherichia coli prlC gene has been cloned into the pET28a prokaryotic expression vector. The recombinant fusion protein was produced mostly in the soluble, active form and the expression level amounted to approximately 70% of total protein. The recombinant proteinase was efficiently adsorbed to a resin containing immobilized Ni2+ via its amino terminal fusion hexahistidine tail to give a PrlC proteinase affinity column. The adsorbed fusion proteinase hydrolyzed 4-methylcoumaryl-7-amide of tert-butoxycarbonyl-l-valyl-l-prolyl-l-arginine (Boc-Val-Pro-Arg-NH-Mec), the specific substrate for the trypsin-like proteinase activity of E. coli PrlC. 相似文献
8.
Kisung Ko John L. Norelli Susan K. Brown Herb S. Aldwinckle 《Biotechnology Techniques》1999,13(12):849-857
A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l–1) by Ni2+-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody. 相似文献
9.
Barbara Kroczynska Rengang Zhou Clifford Wood Jan A. Miernyk 《Plant molecular biology》1996,31(3):619-629
The nucleotide sequence of a cDNA clone fromArabidopsis thaliana ecotype Columbia was determined, and the corresponding amino sequence deduced. The open reading frame encodes a protein, AtJ1, of 368 residues with a molecular mass of 41 471 Da and an isoelectric point of 9.2. The predicted sequence contains regions homologous to the J- and cysteine-rich domains ofEscherichia coli DnaJ, but the glycine/phenylalanine-rich region is not present. Based upon Southern analysis,Arabidopsis appears to have a singleatJ1 structural gene. A single species of mRNA, of 1.5 kb, was detected whenArabidopsis poly(A)+ RNA was hybridized with theatJ1 cDNA. The function ofatJ1 was tested by complementation of adnaJ deletion mutant ofE. coli, allowing growth in minimal medium at 44°C. The AtJ1 protein was expressed inE. coli as a fusion with the maltose binding protein. This fusion protein was purified by amylose affinity chromatography, then cleaved by digestion with the activated factor X protease. The recombinant AtJ1 protein was purified to electrophoretic homogeneity.In vitro, recombinant AtJ1 stimulated the ATPase activity of bothE. coli DnaK and maize endosperm cytoplasmic Stress70. The deduced amino acid sequence of AtJ1 contains a potential mitochondrial targeting sequence at the N-terminus. Radioactive recombinant AtJ1 was synthesized inE. coli and purified. When the labeled protein was incubated with intact pea cotyledon mitochondria, it was imported and proteolytically processed in a reaction that depended upon an energized mitochondrial membrane.Abbreviations MBP
maltose binding protein
- PCR
polymerase chain reaction
- Stress70c
the cytosolic member of the 70 kDA family of stress-related proteins 相似文献
10.
Chang Soo Kang Seung-Yeol Son In Seok Bang 《Journal of microbiology (Seoul, Korea)》2008,46(6):656-661
The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine
at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn
(HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector
to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell
proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully
by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography,
and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined
by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial
activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the
amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. 相似文献
11.
Cloning and functional characterization of an O-acetylserine(thiol)lyase-encoding gene in wild soybean (Glycine soja) 总被引:2,自引:0,他引:2
The terminal step of soybean cysteine synthesis is catalyzed by O-acetylserine(thiol)lyase (OAS-TL, EC 2.5.1.47). In this study, we isolated and characterized an OAS-TL gene from a wild soybean material (designated as GsOAS-TL1). GsOAS-TL1 cDNA sequence showed strict conservation at both nucleotide and amino acid levels compared with that from cultivated soybean.
Genomic structure analysis of GsOAS-TL1 indicated that it contained 10 exons and 9 introns in the coding region with conserved exon sizes and intron locations compared
with Arabidopsis thaliana
OAS-TL-like genes. Among the complete GsOAS-TL1 cDNA and three part-deletion fragments, only expression of the full-length cDNA could rescue the NK3 cys−
Escherichia coli auxotroph, which was coherent with the assayed enzyme activity of purified fusion proteins. For RT-PCR analysis in different
wild soybean tissues, GsOAS-TL1 showed lower expression in roots and developing seeds, whereas total OAS-TL activity of corresponding tissues showed significantly
higher level in seeds than other tissues. To our knowledge, this is the first report on cloning and characterization of an
OAS-TL gene from wild soybean. Our results are informative to further elucidate the function and evolution of OAS-TL in soybean. 相似文献
12.
Akihiro Ota 《Molecular and cellular biochemistry》1986,71(1):87-93
Summary A Co2+-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10–3 M KCN and completely by 10–3 M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45°C and 50°C, respectively. The apparent Km value was 6.7 × 10–4 M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.Abbreviations EDTA
Ethylenediaminetetraacetate
- PCMB
pchloromercuribenzoate 相似文献
13.
Dipika G. Roy Todd R. Klaenhammer Hosni M. Hassan 《Molecular & general genetics : MGG》1993,239(1-2):33-40
The Escherichia coli sodA gene encoding the antioxidant enzyme Mn-containing superoxide dismutase (MnSOD), was cloned in the expression vector pMG36e. This vector has a multiple cloning site down-stream of a promoter and Shine-Dalgarno sequences derived from Lactococcus. The protein-coding region of sodA from E. coli was amplified by the polymerase chain reaction, using a thermocycler and Taq DNA polymerase before cloning into pMG36e. When introduced into E. coli, the recombinant plasmid expressed the predicted fusion protein, both in the presence and absence of oxygen. The expression of the fusion protein in E. coli was verified by SOD assays, activity gels and Western blots. The recombinant plasmid was also introduced into Lactococcus lactis, which contains a resident SOD, and into Lactobacillus gasseri, which is devoid of SOD. Transformed lactococci expressed an active SodA fusion protein plus an active hybrid protein composed of subunits of the Lactococcus and the recombinant E. coli enzymes. Transformants of L. gasseri expressed only the fusion SodA protein, which was enzymatically active. 相似文献
14.
15.
Kim MS Seo JS Choi GE Lim SU Chung JK Lee HH 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2004,139(4):681-693
A gene encoding phosphoinositide-specific phospholipase C (PLC), designated ML-PLCδ, was cloned from mud loach (Misgurnus mizolepis) liver. A complete cDNA encoding ML-PLCδ was isolated by screening the cDNA library of mud loach liver and using the 5′-rapid amplification of cDNA ends (RACE) method. The full-length ML-PLCδ gene contains an open reading frame of 2325 base pairs encoding a 774 amino acid protein with a molecular mass of 88,072 Da; this corresponds to the size of the protein expressed in Escherichia coli BL21 (DE3) using pET28a vector. It contains all of the characteristic domains found in mammalian PLCδ isozymes (PH domain, EF-hands, X–Y catalytic region, and a C2 domain). A homology search revealed that ML-PLCδ shares relatively high sequence identity with mammalian PLCδ1 (51–52%) and catfish PLCδ (64%). The recombinant ML-PLCδ protein expressed as a histidine-tagged fusion protein in E. coli was purified to apparent homogeneity by Ni2+-NTA affinity chromatography. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP2) and its activity was Ca2+-dependent, which was similar to mammalian PLCδ isozymes. 相似文献
16.
A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl−1 at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl−1 at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus. 相似文献
17.
乌桕是一种重要的木本油料树种。SAD(stearoyl-acyl ACP desaturase)是油料植物中将饱和脂肪酸转变成不饱和脂肪酸的一种关键脱氢酶。为了进一步揭示乌桕SsSAD的功能,该研究在大肠杆菌中表达了该蛋白。结果表明:(1)通过RT-PCR的方法从乌桕种子中克隆出了SsSAD基因编码区全长序列,并将其克隆到低温诱导的原核表达载体pCold TF上,构建原核重组表达载体pCold TF/SsSAD,转化大肠杆菌BL 21star(DE3)并获得原核表达工程菌株。(2)通过IPTG法低温诱导表达融合蛋白。该重组质粒在大肠杆菌中得到了高效表达,融合蛋白分子质量约为101kD,且在上清液和包涵体中均有表达,可溶性部分经亲和层析纯化和Western blotting检测证实获得了重组蛋白,上述结果为进一步研究乌桕SsSAD的结构和功能奠定了基础。 相似文献
18.
Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth in mammalian species, and its activity is inhibited
by MSTN prodomain, the N-terminal part of proMSTN cleaved during post-translational MSTN processing. In fish, MSTN also appears
to suppress fish muscle growth with its activity being inhibited by prodomain. The objective of this study was to produce
bioactive MSTN-1 prodomain of rockfish (S. schlegeli), a commercial aquaculture species in East Asia, in E. coli using maltose binding protein (MBP) as a fusion partner. Rockfish MSTN-1 prodomain (sMSTN1pro) cDNA was cloned into the pMALc2x
vector, and proteins (MBP-sMSTN1pro) were expressed in Rosetta-gami 2(DE3)pLysS cells by IPTG induction. The MBP-sMSTN1pro
was expressed in soluble forms, and affinity purified using amylose resin. The affinity purified MBP-sMSTN1pro suppressed
MSTN activity in vitro. The results suggest that MBP is probably a useful fusion partner in producing bioactive MSTN prodomains
of various animal species in E. coli. 相似文献
19.
In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene. We previously cloned the soybean (Glycine max cv. Century) DapA gene in Escherichia coli to express functional soybean DS protein. Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine. Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications. When expressed in E. coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM. 相似文献
20.
Muhammad Nur Cahyanto Hiroko Kawasaki Kazuhito Fujiyama Tatsuji Seki 《World journal of microbiology & biotechnology》2006,22(4):409-416
Summary The lysine biosynthetic genes asd, dapA, and dapB, encoding aspartate semialdehyde dehydrogenase (ASADH), dihydrodipicolinate synthase (DHPS), and dihydrodipicolinate reductase
(DHPR), respectively, have been cloned from Lactobacillus plantarum IAM 12477 by heterologous complementation to Escherichia coli mutants. The amino acid sequences of the cloned genes showed considerable similarities to the corresponding protein from
other gram-positive bacteria. We identified the amino acids that correspond to key catalytic residues of ASADH, DHPS, and
DHPR and found them to be conserved in the protein from L. plantarum. ASADH, DHPS, and DHPR activity was detected in the cell extracts of E. coli mutant harboring each gene, indicating that the cloned genes were functionally expressed in E. coli. The regulation of ASADH, DHPS, and DHPR were studied in the cell extracts of both the E.␣coli mutant harboring the gene and L. plantarum; however, those enzymes were found not to be regulated by the end products of the pathway.
This paper represents a portion of the thesis submitted by M. N. Cahyanto to Osaka University as partial fulfillment of the
requirements for the PhD degree. 相似文献