scholarly journals Cloning and functional expression of B chains of β-bungarotoxins from Bungarus multicinctus (Taiwan banded krait)

1998 ◽  
Vol 334 (1) ◽  
pp. 87-92 ◽  
Author(s):  
Pei-Fung WU ◽  
Sheng-Nan WU ◽  
Chun-Chang CHANG ◽  
Long-Sen CHANG
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Functional Expression ◽  
Coli Strain ◽  
Amino Acid Sequences ◽  
Protein Sequencing ◽  
K Channel ◽  
Venom Glands ◽  
A Chain

The cDNA species encoding the B chains (B1 and B2) of β-bungarotoxins (β-Bgt) were constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus (Taiwan banded krait). The deduced amino acid sequences of the B chains were different from those determined previously by a protein sequencing technique. One additional Arg residue is inserted between Val-19 and Arg-20 of the B1 chain. Similarly the insertion of one additional Val residue between Val-19 and Arg-20 of the B2 chain is noted. Thus the B chains should comprise 61 amino acid residues. Moreover, the residues at positions 44–46 are Gly-Asn-His, in contrast with a previous result showing the sequence His-Gly-Asn. Instead of Asp, the residues at positions 41 and 43 are Asn. The B chain was subcloned into the expression vector pET-32a(+) and transformed into Escherichia coli strain BL21(DE3). The recombinant B chain was expressed as a fusion protein and purified on a His-Bind resin column. The yield of affinity-purified fusion protein was increased markedly by replacing Cys-55 of the B chain with Ser. However, the isolated B(C55S) chain became insoluble in aqueous solution after removal of the fused protein from the affinity-purified product, suggesting that protein–protein interactions might be crucial for stabilizing the structure of the B chain. The B(C55S) chain fusion protein showed activity in blocking the voltage-dependent K+ channel, but did not inhibit the binding of β-Bgt to synaptosomal membranes. These results, together with the finding that modification of His-48 of the A chain of β-Bgt caused a marked decrease in the ability to bind toxin to its acceptor proteins, suggest that the B chain is involved in the K+ channel blocking action observed with β-Bgt, and that the binding of β-Bgt to neuronal receptors is not heavily dependent on the B chain.

scholarly journals The Fine Structure of the Sensory Epithelium of the Vomeronasal Organ in Suckling Rats

1971 ◽  
Vol 24 (3) ◽  
pp. 765 ◽  
Author(s):  
Jean E Kratzing
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Vomeronasal Organ ◽  
Sensory Epithelium ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Tryptic Peptides ◽  
Special Procedure ◽  
A Chain

The amino acid sequence of the a-chain of haemoglobin from M. giganteus has been determined. The soluble peptides formed by tryptic digestion were isolated by gel filtration, ion-exchange chromatography, paper ionophoresis, and chromatography. The amino acid sequences were determined by the "dansyl"Edman procedure. Incomplete hydrolysis of one bond resulted in a large insolublecore peptide containing 40 amino acid residues. The sequence of this peptide was deduced from the sequences of smaller peptides resulting from further digestion with thermolysin and papain. Maleylation of the a-globin before tryptic digestion gave three large fragments which assisted in assigning tryptic peptides to specific areas of the molecule. A special procedure involving maleylation of a chymotryptic digest of globin was used to isolate peptides containing arginine which provided overlap sequences of tryptic peptides

scholarly journals The G9a gene in the human major histocompatibility complex encodes a novel protein containing ankyrin-like repeats

1993 ◽  
Vol 290 (3) ◽  
pp. 811-818 ◽  
Author(s):  
C M Milner ◽  
R D Campbell
Keyword(s):  
Major Histocompatibility Complex ◽  
Amino Acid ◽  
Fusion Protein ◽  
Protein Interactions ◽  
Terminal Region ◽  
Major Histocompatibility ◽  
Class Iii ◽  
Intracellular Protein ◽  
Human Major Histocompatibility Complex ◽  
Histocompatibility Complex

The class III region of the human major histocompatibility complex spans approx. 1.1 Mbp on the short arm of chromosome 6 and is known to contain at least 36 genes. The complete nucleotide sequence of a 3.4 kb mRNA from one of these genes, G9a (or BAT8), has been determined from cDNA and genomic DNA clones. The single-copy G9a gene encodes a protein product of 1001 amino acids with a predicted molecular mass of 111,518 Da. The C-terminal region (residues 730-999) of the G9a protein has been expressed in Escherichia coli as a fusion protein with the 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26). The fusion protein has been used to raise antisera which, in Western-blot analysis, cross-react specifically with an intracellular protein of approx. 98 kDa. The function of the G9a protein is unknown. However, comparison of the derived amino acid sequence of G9a with the protein databases has revealed interesting similarities with a number of other proteins. The C-terminal region of G9a is 35% identical with a 149 amino acid segment of the Drosophila trithorax protein. In addition the G9a protein has been shown to contain six contiguous copies of a 33-amino acid repeat. This repeat, originally identified in the Notch protein of Drosophila and known as the cdc10/SW16 or ANK repeat, is also found in a number of other human proteins and may be involved in intracellular protein-protein interactions.

scholarly journals Protein structure and gene cloning of Syncephalastrum racemosum nuclease

1999 ◽  
Vol 339 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Heng-Chien HO ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Direct Sequencing ◽  
Coli Strain ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Computer Search ◽  
Degenerate Primers ◽  
Syncephalastrum Racemosum

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

scholarly journals Cloning and Characterization of the xyl1 Gene, Encoding an NADH-Preferring Xylose Reductase from Candida parapsilosis, and Its Functional Expression in Candida tropicalis

2003 ◽  
Vol 69 (10) ◽  
pp. 6179-6188 ◽  
Author(s):  
Jung-Kul Lee ◽  
Bong-Seong Koo ◽  
Sang-Yong Kim
Keyword(s):  
Amino Acid ◽  
Candida Tropicalis ◽  
Candida Parapsilosis ◽  
Xylose Reductase ◽  
Wild Strain ◽  
Catalytic Efficiency ◽  
Functional Expression ◽  
Amino Acid Sequences ◽  
Gene Encoding ◽  
Coenzyme Specificity

ABSTRACT Xylose reductase (XR) is a key enzyme in d-xylose metabolism, catalyzing the reduction of d-xylose to xylitol. An NADH-preferring XR was purified to homogeneity from Candida parapsilosis KFCC-10875, and the xyl1 gene encoding a 324-amino-acid polypeptide with a molecular mass of 36,629 Da was subsequently isolated using internal amino acid sequences and 5′ and 3′ rapid amplification of cDNA ends. The C. parapsilosis XR showed high catalytic efficiency (k cat/Km = 1.46 s−1 mM−1) for d-xylose and showed unusual coenzyme specificity, with greater catalytic efficiency with NADH (k cat/Km = 1.39 × 104 s−1 mM−1) than with NADPH (k cat/Km = 1.27 × 102 s−1 mM−1), unlike all other aldose reductases characterized. Studies of initial velocity and product inhibition suggest that the reaction proceeds via a sequentially ordered Bi Bi mechanism, which is typical of XRs. Candida tropicalis KFCC-10960 has been reported to have the highest xylitol production yield and rate. It has been suggested, however, that NADPH-dependent XRs, including the XR of C. tropicalis, are limited by the coenzyme availability and thus limit the production of xylitol. The C. parapsilosis xyl1 gene was placed under the control of an alcohol dehydrogenase promoter and integrated into the genome of C. tropicalis. The resulting recombinant yeast, C. tropicalis BN-1, showed higher yield and productivity (by 5 and 25%, respectively) than the wild strain and lower production of by-products, thus facilitating the purification process. The XRs partially purified from C. tropicalis BN-1 exhibited dual coenzyme specificity for both NADH and NADPH, indicating the functional expression of the C. parapsilosis xyl1 gene in C. tropicalis BN-1. This is the first report of the cloning of an xyl1 gene encoding an NADH-preferring XR and its functional expression in C. tropicalis, a yeast currently used for industrial production of xylitol.

Characterization, chromosomal mapping, and expression of different ubiquitin fusion protein genes in tissues from control and heat-shocked maize seedlings

1996 ◽  
Vol 74 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Ling Liu ◽  
J. Roger H. Frappier ◽  
Karen d'Ailly ◽  
Burr G. Atkinson ◽  
Daniel S. Maillet ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Fusion Protein ◽  
Specific Protein ◽  
Amino Acid Sequences ◽  
Chromosomal Mapping ◽  
Repeat Units ◽  
Acid Protein ◽  
Specific Mrnas ◽  
Shock Proteins

Organisms possess at least two multigene families of ubiquitins: the polyubiquitins, with few to several repeat units, which encode a ubiquitin monomer, and the ubiquitin fusion (or extension) protein genes, which encode a single ubiquitin monomer and a specific protein. This report provides details about two ubiquitin fusion protein genes in maize referred to as MubG7 (uwo 1) and MubG10 (uwo 2). Each has one nearly identical ubiquitin coding unit fused without an intervening nucleotide to an unrelated, 237-nucleotide sequence that encodes for a 79 amino acid protein. The derived amino acid sequences of the two fusion proteins show that they differ by five amino acids (substitution by either a serine or threonine). MubG7 maps to chromosome 8L162 and MubG10 maps to chromosome 1L131. Analyses of the role(s) of these genes in response to heat shock (1 h at 42.5 °C) reveal that the level of these fusion protein mRNAs in the radicles or plumules from 2-day-old seedlings does not change; however, heat shock does cause a marked reduction in the accumulation of these same gene-specific mRNAs in the radicles and plumules of 5-day-old seedlings. These data confirm the suggestion from our earlier work that there is precise modulation, in a gene-specific manner, of the response to developmental as well as environmental signals.Key words: ubiquitin, ubiquitin extension (or fusion) protein, maize, heat shock, heat shock proteins, gene expression, chromosome map.

scholarly journals Studies on Monotreme Proteins. V. Amino Acid Sequence of the a-Chain of Haemoglobin From the Platypus, Ornithorhynchus anatinus

1974 ◽  
Vol 27 (6) ◽  
pp. 591 ◽  
Author(s):  
RG Whittaker ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Complete Amino Acid Sequence ◽  
Ornithorhynchus Anatinus ◽  
A Chain

Blood from the platypus contained three haemoglobins which were separated by chromatography on DEAE-Sephadex. The major component, Hb-I, was converted to globin and fractionated into the oc-and p-chains by chromatography on eM-cellulose in 8M urea-thiol buffers, and the complete amino acid sequence of the 141 residues of the oc-chain were determined. Peptides derived from the oc-chain by tryptic digestion were isolated by paper ionophoresis and chromatography. The amino acid sequences were determined by the dansyl-Edman procedure or by further digestion with other enzymes.

scholarly journals Haemoglobins of the Shark, Heterodontus Portusjacksoni II. Amino Acid Sequence of the a-Chain

1976 ◽  
Vol 29 (2) ◽  
pp. 73 ◽  
Author(s):  
AR Nash ◽  
WK Fisher ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Paper Chromatography ◽  
Cyanogen Bromide ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Amino Terminal ◽  
Core Peptide ◽  
A Chain

The amino acid sequence of the a-chain of the principal haemoglobin from the shark, H. portusjacksoni has been determined. The chain has 148 residues and is acetylated at the amino terminal. The soluble peptides obtained by tryptic and chymotryptic digestion of the protein or its cyanogen bromide fragments were isolated by gel filtration, paper ionophoresis and paper chromatography. The amino acid sequences were determined by the dansyl-Edman procedure. The insoluble 'core' peptide from the tryptic digestion contained 34 residues and required cleavage by several proteases before the sequence was established. Compared with human a-chain there are 88 amino acid differences including the additional seven residues which appear on the amino terminal of the shark chain. There is also one deletion and one insertion. The chain contains no tryptophan but has four cysteinyl residues which is the highest number of such residues recorded for a vertebrate globin.

In Silico Design of Fusion Toxin DT389GCSF and a Comparative Study

2020 ◽  
Vol 16 (3) ◽  
pp. 238-244 ◽  
Author(s):  
Maryam G. Siahmazgi ◽  
Mohammad A.N. Khalili ◽  
Fathollah Ahmadpour ◽  
Sirus Khodadadi ◽  
Mehdi Zeinoddini
Keyword(s):  
Fusion Protein ◽  
Protein Interactions ◽  
In Silico ◽  
Stimulate Factor ◽  
Web Server ◽  
Colony Stimulate Factor ◽  
Protein Docking ◽  
Amino Acid Sequences ◽  
Fusion Toxin ◽  
Normal Tissues

Background: Chemotherapy and radiotherapy have negative effects on normal tissues and they are very expensive and lengthy treatments. These disadvantages have recently attracted researchers to the new methods that specifically affect cancerous tissues and have lower damage to normal tissues. One of these methods is the use of intelligent recombinant fusion toxin. The fusion toxin DTGCSF, which consists of linked Diphtheria Toxin (DT) and Granulocyte Colony Stimulate Factor (GCSF), was first studied by Chadwick et al. in 1993 where HATPL linker provided the linking sequence between GCSF and the 486 amino acid sequences of DT. Methods: In this study, the fusion toxin DT389GCSF is evaluated for functional structure in silico. With the idea of the commercial fusion toxin of Ontak, the DT in this fusion protein is designed incomplete for 389 amino acids and is linked to the beginning of the GCSF cytokine via the SG4SM linker (DT389GCSF). The affinity of the DT389GCSF as a ligand with GCSF-R as receptor was compared with DT486GCSF as a ligand with GCSF-R as receptor. Both DT486GCSF and its receptor GCSF-R have been modeled by Easy Modeler2 software. Our fusion protein (DT389GCSF) and GCSF-R are modeled through Modeller software; all of the structures were confirmed by server MDWEB and VMD software. Then, the interaction studies between two proteins are done using protein-protein docking (HADDOCK 2.2 web server) for both the fusion protein in this study and DT486GCSF. Results: The HADDOCK results demonstrate that the interaction of DT389GCSF with GCSF-R is very different and has a more powerful interaction than DT486GCSF with GCSF-R. Conclusion: HADDOCK web server is operative tools for evaluation of protein–protein interactions, therefore, in silico study of DT389GCSF will help with studying the function and the structure of these molecules. Moreover, DT389GCSF may have important new therapeutic applications.

Non-neuronal 210 × 10(3) Mr microtubule-associated protein (MAP4) contains a domain homologous to the microtubule-binding domains of neuronal MAP2 and tau

1991 ◽  
Vol 98 (1) ◽  
pp. 27-36
Author(s):  
S.J. Chapin ◽  
J.C. Bulinski
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Hela Cell ◽  
Fusion Proteins ◽  
Fetal Brain ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Projection Domain

A polyclonal antiserum raised against a HeLa cell microtubule-associated protein of Mr 210,000 (210 kD MAP or MAP4), an abundant non-neuronal MAP, was used to isolate cDNA clones encoding MAP4 from a human fetal brain lambda gt11 cDNA expression library. The largest of these clones, pMAP4.245, contains an insert of 4.1 kb and encodes a 245 kD beta-galactosidase fusion protein. Evidence that pMAP4.245 encodes MAP4 sequences includes immunoabsorption of MAP4 antibodies with the pMAP4.245 fusion protein, as well as identity of protein sequences obtained from HeLa 210 kD MAP4 with amino acid sequences encoded by pMAP4.245. The MAP4.245 cDNA hybridizes to several large (approximately 6–9 kb) transcripts on Northern blots of HeLa cell RNA. DNA sequencing of overlapping MAP4 cDNA clones revealed a long open reading frame containing a C-terminal region with three imperfect 18-amino acid repeats; this region is homologous to a motif present in the microtubule (MT)-binding domain of two prominent neuronal MAPs, MAP2 and tau. The pMAP4.245 sequence also encoded a series of unrelated repeats, located in the MAP's projection domain, N-terminal to the MT-binding domain. MAP4.245 fusion proteins bound to MTs in vitro, while fusion proteins that contained only the projection domain repeats failed to bind specifically to MTs. Thus, the major human non-neuronal MAP resembles two neuronal MAPs in its MT-binding domain, while most of the molecule has sequences, and presumably functions, distinct from those of the neuronal MAPs.

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