共查询到20条相似文献,搜索用时 328 毫秒
1.
A cDNA encoding UDP-glucose: formononetin 7-O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L. cell-suspension cultures using probes from Scutellaria baicalensis UDP-glucose: flavonoid 7-O-glucosyltransferase. The open reading frame of the UGT73F1 cDNA encodes a 441-amino-acid protein with a predicted molecular mass of 48.7 kDa. The deduced amino acid sequence showed that the protein is related to the stress-inducible glucosyltransferases. UGT73F1 mRNA was not detected in untreated G. echinata cultures but was transiently induced by treatment with yeast extract. Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli and purified to near homogeneity by nickel chelate chromatography. The purified recombinant enzyme was selective for isoflavonoid, formononetin and daidzein as substrates, while flavonoids and various tested non-flavonoid compounds were poor substrates.Abbreviations GT
UDP-glycosyltransferase
- rUGT73F1
recombinant UGT73F1
- UBGT: UDP-glucose:
baicalein 7-O-glucosyltransferase
The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB098614. 相似文献
2.
Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase
A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric
point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate,
4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity
to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters).
Received: 14 July 2000 / Accepted: 8 August 2000 相似文献
3.
Two isoforms of chalcone synthase (CHS) were isolated from cDNA libraries derived from UV-A-irradiated anthocyanin-accumulating
(DCb) and non-accumulating (DCs) cell cultures of carrot (Daucus carota L.). The clones designated as DcCHS1, which were present only in the DCb library, had a deduced primary sequence of 389 amino
acids and an expected molecular mass of 42.7 kDa, and seem to be alleles of those cloned by Ozeki et al. (1993). The second
isoform (DcCHS2) was present in both libraries. It had the highest degree of similarity (97.7%) to parsley CHS over all 397
amino acids. The expected molecular mass of the corresponding protein was 43.6 kDa. Results obtained from Southern blot analysis
indicated the existence of at least two CHS genes in carrot. A transient enhancement of the DcCHS1 mRNA level after continuous
irradiation with UV-A light could only be observed in anthocyanin-accumulating cultures, whereas an increase in DcCHS2 mRNA
was seen in both cell lines. The maximum accumulation of CHS mRNA occurred 48 h after the onset of UV-A irradiation. In the
European wild carrot the accumulation of DcCHS1 mRNA was restricted to the red central flowers, whereas the DcCHS2 mRNA was
detectable in all red and white petals, as well as leaves, but was absent in stems and roots. The expression of DcCHS1 was
restricted to anthocyanin-accumulating cells or organs. The heterologous expression of both cDNAs in Escherichia coli resulted in immunostainable bands of different sizes on the Western blot and high levels of catalytic CHS activity.
Received: 2 September 1999 / Accepted: 30 November 1999 相似文献
4.
Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta
210: 34–40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30–40% amino acid identity
with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial
cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated.
The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic
cyanobacteria.
Received: 5 January 2000 / Accepted: 7 March 2000 相似文献
5.
3-Deoxyanthocyanins are rare anthocyanin pigments produced by some mosses, ferns, and higher plants. The enzymes and genes
responsible for biosynthesis of 3-deoxyanthocyanins have not been well characterized. We identified a novel gene encoding
UDP-glucose:3-deoxyanthocyanidin 5-O-glucosyltransferase (dA5GT) from Sinningia cardinalis, which accumulates abundant 3-deoxyanthocyanins in its petals. Five candidate genes (ScUGT1 to ScUGT5) were isolated from an S. cardinalis flower cDNA by degenerate PCR targeted for the UGT88 clade. ScUGT1, ScUGT3, and ScUGT5 exhibited 45–47% identity with rose
anthocyanidin 5,3-O-glucosyltransferase, which catalyzes glucosylation at the 5- and 3-position of 3-hydroxyanthocyanidin. Based on its temporal
and spatial gene expression patterns, and enzymatic activity assays of the recombinant protein, ScUGT5 was screened as a dA5GT
candidate. Recombinant ScUGT5 protein expressed in Escherichia coli was used to analyze the detailed enzymatic properties. The results demonstrated that ScUGT5 specifically transferred a glucosyl
moiety to 3-deoxyanthocyanidins in the presence of UDP-glucose, but not to other flavonoid compounds, such as 3-hydroxyanthocyanidins,
flavones, flavonols, or flavanones. 相似文献
6.
CD84 is a member of the immunoglobulin gene superfamily (IgSF) with two Ig-like domains expressed primarily on B lymphocytes
and macrophages. Here we describe the cloning of the mouse homologue of human CD84. Mouse CD84 cDNA clones were isolated from
a macrophage library. The nucleotide sequence of mouse CD84 was shown to include an open reading frame encoding a putative
329 amino acid protein composed of a 21 amino acid leader peptide, two extracellular immunoglobulin (Ig)-like domains, a hydrophobic
transmembrane region, and an 87 amino acid cytoplasmic domain. Mouse CD84 shares 57.3% amino acid sequence identity (88.7%,
considering conservative amino acid substitutions) with the human homologue. Chromosome localization studies mapped the mouse
CD84 gene to distal chromosome 1 adjacent to the gene for Ly-9, placing it close to the region where other members of the CD2 IgSF (CD48 and 2B4) have been mapped. Northern blot analysis revealed that the expression of mouse CD84 was predominantly restricted to hematopoietic
tissues. Two species of mRNA of 3.6 kilobases (kb) and 1.5 kb were observed. The finding that the pattern of expression was
restricted to the hematopoietic system and the conserved sequence of the mouse CD84 homologue suggests that the function of
the CD84 glycoprotein may be similar in humans and mice.
Received: 1 July 1998 / Revised: 31 August 1998 相似文献
7.
B. Jongsareejit R. N. Z. A. Rahman S. Fujiwara T. Imanaka 《Molecular & general genetics : MGG》1997,254(6):635-642
The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that
are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an
FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the
upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed
in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer
of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that
KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis
of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13).
The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending
on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C).
Received: 30 October 1996 / Accepted: 13 January 1997 相似文献
8.
The gene lccK encoding a laccase of the white-rot basidiomycete Pleurotus ostreatus wild-type strain collected in Japan has been cloned, sequenced, and characterized. The isolated gene consists of 2929 bp
with the coding region interrupted by 19 introns and flanked by an upstream region in which putative CAAT and TATA elements
were identified. Two putative N-glycosylation sites and four putative copper-binding sites found in other fungal laccase are
conserved in lccK. The cDNA contains an open reading frame of 1599 bp and the gene encodes 533 amino acids preceded by a signal peptide of
23 amino acids. The nucleotide sequence of the lccK cDNA showed high homology with those of laccases of other basidiomycetes.
Received: August 22, 2002 / Accepted: October 9, 2002
Present address: Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo-nakano, Akita 010-0195, Japan
Correspondence to:K. Okamoto 相似文献
9.
Summary. The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it
contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated
clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions
significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional
specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically
important for full enzyme activity. 相似文献
10.
A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum,
where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has
been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction
using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da).
In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature
OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Received: 29 July 1999 / Accepted: 25 August 1999 相似文献
11.
Summary. Protein L4 from the thermophilic bacterium Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells and purified under native conditions by using ion exchange chromatography. Although it’s known strong binding to RNA
(23S rRNA as well as mRNA) the yield of the purified protein was 6 mg per 10 g of cells and it is similar to that referred
for Thermotoga maritima L4 ribosomal protein.
In addition, E. coli cells harboring the wild type Thermus thermophilus L4 (wtTthL4) ribosomal protein as well as its mutant having changed the highly conserved glutamic acid 56 by alanine (TthL4-Ala
56) were incorporated into E. coli ribosomes after transformation of the host cells with the recombined vector. The cells having incorporated the mutant TthL4-Ala56
are more sensitive against erythromycin related to that containing the wtTthL4 protein.
The resistance to the drug indicates that the mutated amino acid Glu56 is probably critical for the local ribosomal conformation
and that its mutation induces conformational disturbances that are “transferred” to the entrance of the major exit tunnel,
the place where the drug does bind. 相似文献
12.
13.
Uridine 5′-diphosphoglucose:betanidin 5-O- and 6-O-glucosyltransferases (5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific formation of betanin (betanidin 5-O-β-glucoside) and gomphrenin I (betanidin 6-O-β-glucoside), respectively. Both enzymes were purified to near homogeneity from cell-suspension cultures of Dorotheanthus bellidiformis, the 5-GT by classical chromatographic techniques and the 6-GT by affinity dye-ligand chromatography using UDP-glucose as
eluent. Data obtained with highly purified enzymes indicate that 5-GT and 6-GT catalyze the indiscriminate transfer of glucose
from UDP-glucose to hydroxyl groups of betanidin, flavonols, anthocyanidins and flavones, but discriminate between individual
hydroxyl groups of the respective acceptor compounds. The 5-GT catalyzes the transfer of glucose to the C-4′ hydroxyl group
of quercetin as its best substrate, and the 6-GT to the C-3 hydroxyl group of cyanidin as its best substrate. Both enzymes
also catalyze the formation of the respective 7-O-glucosides, but to a minor extent. Although the enzymes were not isolated to homogeneity, chromatographic, electrophoretic
and kinetic properties proved that the respective enzyme activities were based on the presence of single enzymes, i.e. 5-GT
and 6-GT. The N terminus of the 6-GT revealed high sequence identity to a proposed UDP-glucose:flavonol 3-O-glucosyltransferase (UF3GT) of Manihot esculenta. In addition to the 5-GT and 6-GT, we isolated a UF3GT from D. bellidiformis cell cultures that preferentially accepted myricetin and quercetin, but was inactive with betanidin. The same result was
obtained with a UF3GT from Antirrhinum majus and a flavonol 4′-O-glucosyltransferase from Allium cepa. Based on these results, the main question to be addressed reads: Are the characteristics of the 5-GT and 6-GT indicative
of their phylogenetic relationship with flavonoid glucosyltransferases?
Received: 11 February 1997 / Accepted: 18 April 1997 相似文献
14.
Mato Masami; Ozeki Yoshihiro; Itoh Yoshio; Higeta Daisuke; Yoshitama Kunijiro; Teramoto Susumu; Aida Ryutaro; Ishikura Nariyuki; Shibata Michio 《Plant & cell physiology》1998,39(11):1145-1155
Four cDNA clones were isolated from Vigna mungo seedlings bythe screening with cDNA encoding UDP-glu-cose:flavonoid 3-0-glucosyltransferase(UF3GT) of Antirrhinum majus as a probe; the product of thegene corresponding to one cDNA was more highly expressed inthe first simple leaves than in stems. Nucleotide sequence analysisrevealed 1,691 bp (including 326 bp non-reading) containingan open reading frame of 455 amino acids. The deduced aminoacid sequence showed 42% and 23% identity with those of A. majusUDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) and Petuniahybrida UDP-rhamnose:anthocyanidin 3-0-glucoside rhamnosyltrans-ferase(RT), respectively. One region of the cDNA (amino acids 325to 387) showed similarity to ceramide UDP-galac-tosyltransferasesof mice, rats and humans. A crude extract from Escherichia coli,in which the protein was expressed from the cDNA, showed highUF3GaT activity but low UF3GT activity, and was similar in Km,optimal pH and substrate specificity to UF3GaT from V. mungo.We conclude that we have obtained UDP-galactose:flavonoid 3-0-galactosyltransferase(UF3GaT) cDNA from V. mungo.
4 Deceased. 相似文献
15.
A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in
the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated
molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted
amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active
at pH 7 and a temperature of 40° C.
Received: 6 February 1998 / Accepted: 6 November 1998 相似文献
16.
High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode 总被引:8,自引:0,他引:8
Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective
medium containing 200 μg ml−1 kanamycin and 500 μg ml−1 carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene
incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the
Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for
the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode
(Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could
complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for
testing genes that might impart resistance to soybean cyst nematode.
Received: 13 July 1999 / Accepted: 8 August 1999 相似文献
17.
A. Phongdara A. Merckelbach P. Keup G. Gellissen C. P. Hollenberg 《Applied microbiology and biotechnology》1998,50(1):77-84
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA
fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with
a calculated M
r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide
sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved
in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998 相似文献
18.
A cDNA fragment encoding a Lupinus albus. L. class-III chitinase, IF3, was isolated, using a cDNA probe from Cucumis sativus L., by in-situ plaque hybridization from a cDNA library constructed in the Uni-ZAP XR vector, with mRNAs isolated from mature
lupin leaves. The cDNA had a coding sequence of 293 amino acids including a 27-residue N-terminal signal peptide. A class-III
chitinase gene was detected by Southern analysis in the L. albus genome. Western blotting experiments showed that the IF3 protein was constitutively present during seed development and in
all the studied vegetative lupin organs (i.e., roots, hypocotyls and leaves) at two growth stages (7- and 20-d-old plants).
Accumulation of both the IF3 mRNA and IF3 protein was triggered by salicylic acid treatment as well as by abiotic (UV-C light
and wounding) and biotic stress conditions (Colletotrichum gloeosporioides infection). In necrotic leaves, IF3 chitinase mRNA was present at a higher level than that of another mRNA encoding a pathogenesis-related
(PR) protein from L. albus (a PR-10) and that of the rRNAs. We suggest that one role of the IF3 chitinase could be in the defense of the plant against
fungal infection, though our results do not exclude other functions for this protein.
Received: 15 March 1999 / Accepted: 12 July 1999 相似文献
19.
Biosynthesis of the (1,3)-β-d-glucan (curdlan) in Agrobacterium sp., is believed to proceed by the repetitive addition of glucosyl residues from UDP-glucose by a membrane-embedded curdlan
synthase (CrdS) [UDP-glucose: (1,3)-β-d-glucan 3-β-d-glucosyltransferase; EC 2.4.1.34]. The catalytic module of CrdS (cm-CrdS) was expressed in good yield from a cDNA encoding
cm-CrdS cloned into the pET-32a(+) vector, containing a coding region for thioredoxin, and from the Champion™ pET SUMO system
that possesses a coding region of a small ubiquitin-related modifier (SUMO) partner protein. The two DNA fusions, designated
pET-32a_cm-CrdS and SUMO_cm-CrdS were expressed as chimeric proteins. High yields of inclusion bodies were produced in E. coli and these could be refolded to form soluble proteins, using a range of buffers and non-detergent sulfobetaines. A purification
protocol was developed, which afforded a one-step on-column refolding and simultaneous purification of the recombinant 6xHis-tagged
SUMO_cm-CrdS protein. The latter protein was digested by a specific protease to yield intact cm-CrdS in high yields. The refolded
SUMO_cm-CrdS protein did not exhibit curdlan synthase activity, but showed a circular dischroism spectrum, which had an α/β-type-like
conformation. Amino acid sequences of tryptic fragments of the SUMO_cm-CrdS fusion and free cm-CrdS proteins, determined by
MALDI/TOF confirmed that the full-length proteins were synthesized by E. coli, and that no alterations in amino acid sequences occurred. A three-dimensional model of cm-CrdS predicted the juxtaposition
of highly conserved aspartates D156, D208, D210 and D304, and the QRTRW motif, which are likely to play roles in donor and
acceptor substrate binding and catalysis. 相似文献
20.
Interleukin-5 (IL-5) is thought to be a key cytokine in allergic inflammation. Pig IL-5 was cloned, sequenced, and expressed
to enable us to study of the biological role of IL-5 in pigs used in a model for allergen-induced late-phase reactions. These
pigs were sensitized to proteins extracted from Ascaris suum, resulting in hypersensitivity to this antigen in both the skin and airways, and a slight blood eosinophilia. Peripheral
blood mononuclear cells from antigen-sensitized pigs were isolated and polyclonally stimulated. Total RNA was extracted and
reverse transcribed into cDNA. IL-5 primers based on the cow IL-5 cDNA sequence were used to obtain an initial polymerase
chain reaction product. 3′ rapid amplification of cDNA ends (3′RACE) and 5′RACE procedures were applied to identify the 3′
and 5′ ends, respectively. The full-length pig IL-5 cDNA is 405 base pairs long. Mature pig IL-5 was expressed in Escherichia coli with a His-tag for purification. The IL-5 protein is 115 amino acids long, has an estimated molecular weight of 14 000 M
r
and forms a biologically active homodimer of 28 000 M
r
. Pig IL-5 shows 65% amino acid identity to the human IL-5 sequence and 90, 88, 83, 62, and 61% identity to the cow, sheep,
horse, mouse, and rat counterparts.
Received: 29 June 1999 / Revised: 22 September 1999 相似文献