• Toxicological Research
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• v.17
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• pp.109-115
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• 2001

Abrus agglutinin was purified from the kernels of Abrus precatorius by Sepharose 4B affinity column chromatography followed by Sephadex G-100 gel filtration column chromatography. About 1.25 g of abrus agglutinin was obtained from 1 kg of the kernels. The LD$_{50}$ of abrus agglutinin is 5 mg/kg of body weight, which is less toxic than that of abrin, 20$\mu\textrm{g}$/kg body weight. The amino acid sequence of abrus agglutinin was determined by protein sequencing techniques and deduced from the nucleotide sequence of a cDNA clone encoding full length of abrus agglutinin. There are 258 residues, 2 residues and 267 residues in the A-chain, the linker peptide and the B-chain of abrus agglutinin, respectively. Abrus agglutinin had high homology to abrin-a (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrin and ricin, were also conserved within abrus agglutinin. The protein synthesis inhibitory activity of abrus agglutinin ($IC_{50}$/ = 3.5 nM) was weaker than that of abrin-a (0.05 nM). By molecular modeling followed by site-directed mutagenesis showed that Pro199 of abrus agglutinin A-chain located in amphipathic helix H and corresponding to Asn200 of abrin A-chain, can induce bending of helix H. This bending would presumably affect the binding of abrus agglutinin A-chain to its target sequence GpApGpAp, in the tetraloop structure of 285 r-RNA subunit and this could be one of major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of abrus agglutinin.n.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1098-1102
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• 2009

Gamma-linolenic acid (GLA, C18:3 ${\Delta}^{6,9,12}$) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 ${\Delta}^{9,12}$) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.

• Animal cells and systems
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• v.11 no.2
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• pp.175-182
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• 2007

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.146-152
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• 2004

Ghrelin is an acylated peptide recently identified as the endogenous ligand for the growth hormone (GH) secretagogues receptor 1a (GHS-R1a) and is involved in a novel system for regulating GH release. To understand the long-term effects of ghrelin, here we constructed six myogenic expression vectors containing the cDNA of swine mature ghrelin (pGEM-wt-sGhln, pGEM-wt-hGhln), ghrelin mutant of $Ser^3$ with $Trp^3$ (pGEM-mt-sGhln, pGEM-mt-hGhln) and truncated ghrelin derivative (pGEM-tmtsGhln, pGEM-tmt-hGhln) encompassing the first 7 residues of ghrelin (including $Ser^3$ substituted with $Trp^3$) and adding a basic amino acid, Lys (K) in the C-terminus. The constructs, pGEM-wt-sGhln, pGEM-mt-sGhln and pGEM-tmt-sGhln were linked with the ghrelin leader sequence, while the pGEM-wt-hGhln, pGEM-mt-hGhln and pGEM-tmt-hGhln were linked with a leader sequence from the human growth hormone releasing hormone (hGHRH). Intramuscular injection of 200 ${\mu}g$ pGEM-wt-sGhln or pGEM-tmt-sGhln augmented growth over 3 weeks in normal rats and peaked at day 21 or 14 post-injection respectively, whose body weight gains were on average approximately 6% or 19% heavier over controls. However, other injectable vectors had no such enhanced growth effects. Our results suggested that the efficacy of the ghrelin leader sequence was more effective than that of hGHRH in our system. Moreover, the results indicated that skeletal muscle might have the ability to posttranslationally modify the in vivo expressed ghrelin. And the most strikingly, the short ghrelin analog seems to mimic the biological effects more efficiently when compared with the full-length ghrelin.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.160-166
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• 2008

A DNA fragment containing 2,079 base pairs from Bacillus circulans CGMCC 1416 was cloned using degenerate PCR and inverse PCR. An open reading frame containing 981 bp was identified that encoding 326 amino acids residues, including a putative signal peptide of 31 residues. The deduced amino acid sequence showed the highest identity (68.1%) with $endo-{\beta}-1,4-D-mannanase$ from Bacillus circulans strain K-1 of the glycoside hydrolase family 5 (GH5). The sequence encoding the mature protein was cloned into the pET-22b(+) vector and expressed in Escherichia coli as a recombinant fusion protein containing an N-terminal hexahistidine sequence. The fusion protein was purified by $Ni^{2+}$ affinity chromatography and its hexahistidine tag cleaved to yield a 31-kDa ${\beta}$-mannanase having a specific activity of 481.55U/mg. The optimal activity of the purified protein, MANB48, was at $58^{\circ}C$ and pH 7.6. The hydrolysis product on substrate locust bean gum included a monosaccharide and mainly oligosaccharides. The recombinant MANB48 may be of potential use in the feed industry.

• Parasites, Hosts and Diseases
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• v.51 no.3
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• pp.327-334
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• 2013

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an ${\alpha}$-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.

• Interdisciplinary Bio Central
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• v.1 no.2
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• pp.8.1-8.6
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• 2009

Introduction: It is a challenge to design a protein score function which stabilizes the native structures of many proteins simultaneously. The coarse-grained description of proteins to construct the pairwise-contact score function usually ignores the backbone directionality of protein structures. We propose a new two-body score function which stabilizes all native states of 1,006 proteins simultaneously. This two-body score function differs from the usual pairwise-contact functions in that it considers two adjacent amino acids at two ends of each peptide bond with the backbone directionality from the N-terminal to the C-terminal. The score is a corresponding propensity for a directional alignment of two adjacent amino acids with their local environments. Results and Discussion: We show that the construction of a directional adjacency-score function was achieved using 1,006 training proteins with the sequence homology less than 30%, which include all representatives of different protein classes. After parameterizing the local environments of amino acids into 9 categories depending on three secondary structures and three kinds of hydrophobicity of amino acids, the 32,400 adjacency-scores of amino acids could be determined by the perceptron learning and the protein threading. These could stabilize simultaneously all native folds of 1,006 training proteins. When these parameters are tested on the new distinct 382 proteins with the sequence homology less than 90%, 371 (97.1%) proteins could recognize their native folds. We also showed using these parameters that the retro sequence of the SH3 domain, the B domain of Staphylococcal protein A, and the B1 domain of Streptococcal protein G could not be stabilized to fold, which agrees with the experimental evidence.

• Journal of Marine Life Science
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• v.1 no.2
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• pp.88-94
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• 2016

Determining the infection history of living organisms is essential for understanding the evolution of infection agents with their host, particularly for key aspects such as immunity. Viruses, which can spread between individuals and often cause disease, have been widely examined. The increasing availability of fish genome sequences has provided specific insights into the diversity and host distribution of retroviruses in fish. The shortspine spurdog (Squalus mitsukurii) is an important elasmobranch species; this medium-sized dogfish typically lives at depths of 100~500 m. However, the retroviral envelope polyprotein in dogfish has not been examined. Thus, the aim of the present study was to identify and analyze the retroviral envelope polyprotein in various tissues of dogfish. The 1334-base pair full-length novel cDNA of dogfish envelope polyprotein (dEnv) was obtained by 3' and 5'-rapid amplification of cDNA end analysis from S. mitsukurii. The open reading frame showed a complete coding sequence of 815 base pairs with a deduced peptide sequence of 183 amino acids that exhibited 34~50% identity with other fish and bird species. It was also expressed according to reverse transcription and real-time polymerase chain reaction in the kidney, liver, intestine, and lung, but not in the gill. This distribution can be assessed by identifying and analyzing endogenous retroviruses in fish, which consists of three main genes: gag, pol and env. Dogfish envelope polyprotein sequence is likely important in evolution and induces rearrangements, altering the regulatory and coding sequences. This is the first report of the identification and molecular characterization of retroviral envelope polyprotein in various tissues of S. mitsukurii.

• Molecules and Cells
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• v.27 no.5
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• pp.547-556
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• 2009

Parathyroid hormone is the most important endocrine regulator of calcium concentration. Its N-terminal fragment (1-34) has sufficient activity for biological function. Recently, site-directed mutagenesis studies demonstrated that substitutions at several positions within shorter analogues (1-14) can enhance the bioactivity to greater than that of PTH (1-34). However, designing the optimal sequence combination is not simple due to complex combinatorial problems. In this study, support vector machines were introduced to predict the biological activity of modified PTH (1-14) analogues using mono-substituted experimental data and to analyze the key physicochemical properties at each position that correlated with bioactivity. This systematic approach can reduce the time and effort needed to obtain desirable molecules by bench experiments and provide useful information in the design of simpler activating molecules.

• Journal of Dairy Science and Biotechnology
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• v.38 no.3
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• pp.142-145
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• 2020

Enterococcus faecalis CAUM157, isolated from raw cow's milk, is a Gram-positive, facultatively anaerobic, and non-spore-forming bacterium capable of inhabiting a wide range of environmental niches. E. faecalis CAUM157 was observed to produce a two-peptide bacteriocin that had a wide range of activity against several pathogens, including Listeria monocytogenes, Staphylococcus aureus, and periodontitis-causing bacteria. The whole genome of E. faecalis CAUM157 was sequenced using the PacBio RS II platform, revealing a genome size of 2,972,812 bp with a G+C ratio of 37.44%, assembled into two contigs. Annotation analysis revealed 2,830 coding sequences, 12 rRNAs, and 61 tRNAs. Further, in silico analysis of the genome identified a single bacteriocin gene cluster.