scholarly journals Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP

2009 ◽  
Vol 191 (15) ◽  
pp. 4996-5009 ◽  
Author(s):  
Dirk Tischler ◽  
Dirk Eulberg ◽  
Silvia Lakner ◽  
Stefan R. Kaschabek ◽  
Willem J. H. van Berkel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Styrene Oxide ◽  
Functional Expression ◽  
Rhodococcus Opacus ◽  
Reading Frame ◽  
Arthrobacter Aurescens ◽  
C Terminus ◽  
Two Component ◽  
Rhodococcus Opacus 1Cp

ABSTRACT Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120.

scholarly journals C-terminal region regulates the functional expression of human noradrenaline transporter splice variants

2006 ◽  
Vol 401 (1) ◽  
pp. 185-195 ◽  
Author(s):  
Chiharu Sogawa ◽  
Kei Kumagai ◽  
Norio Sogawa ◽  
Katsuya Morita ◽  
Toshihiro Dohi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
Splice Variants ◽  
Functional Expression ◽  
Surface Expression ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
C Terminus ◽  
And Function

The NET [noradrenaline (norepinephrine) transporter], an Na+/Cl−-dependent neurotransmitter transporter, has several isoforms produced by alternative splicing in the C-terminal region, each differing in expression and function. We characterized the two major isoforms of human NET, hNET1, which has seven C-terminal amino acids encoded by exon 15, and hNET2, which has 18 amino acids encoded by exon 16, by site-directed mutagenesis in combination with NE (noradrenaline) uptake assays and cell surface biotinylation. Mutants lacking one third or more of the 24 amino acids encoded by exon 14 exhibited neither cell surface expression nor NE uptake activity, with the exception of the mutant lacking the last eight amino acids of hNET2, whose expression and uptake resembled that of the WT (wild-type). A triple alanine replacement of a candidate motif (ENE) in this region mimicked the influences of the truncation. Deletion of either the last three or another four amino acids of the C-terminus encoded by exon 15 in hNET1 reduced the cell surface expression and NE uptake, whereas deletion of all seven residues reduced the transport activity but did not affect the cell surface expression. Replacement of RRR, an endoplasmic reticulum retention motif, by alanine residues in the C-terminus of hNET2 resulted in a similar expression and function compared with the WT, while partly recovering the effects of the mutation of ENE. These findings suggest that in addition to the function of the C-terminus, the common proximal region encoded by exon 14 regulates the functional expression of splice variants, such as hNET1 and hNET2.

scholarly journals Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

2000 ◽  
Vol 182 (22) ◽  
pp. 6440-6450 ◽  
Author(s):  
Jouko Sillanpää ◽  
Beatriz Martínez ◽  
Jenni Antikainen ◽  
Takahiro Toba ◽  
Nisse Kalkkinen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Signal Sequence ◽  
Type I ◽  
Binding Region ◽  
Sequence Comparisons ◽  
Collagen Binding ◽  
Reading Frame ◽  
Lactobacillus Crispatus ◽  
C Terminus

The cbsA gene of Lactobacillus crispatusstrain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. ThecbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three otherLactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsAwhere deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

scholarly journals Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

2019 ◽  
Vol 2 (4) ◽  
pp. 47-55
Author(s):  
Quang Kien Huynh ◽  
An Hoang Nguyen ◽  
Quynh Thi Mong Pham ◽  
Hoan Phuoc Khai Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Clostridium Perfringens ◽  
Field Effect Transistors ◽  
Oral Vaccine ◽  
Soluble Form ◽  
M Cells ◽  
Gastrointestinal Infections ◽  
E Coli ◽  
C Terminus

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

scholarly journals Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Paweł Filipkowski ◽  
Olga Pietrow ◽  
Anna Panek ◽  
Józef Synowiecki
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Deinococcus Radiodurans ◽  
Trehalose Synthase ◽  
Reading Frame ◽  
Metal Affinity ◽  
C Terminus

A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His(6)-tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30°C. DraTreS activity was almost unchanged after 2 h preincubation at 45°C and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50°C. The DraTreS was strongly inhibited by Cu(2+), Hg(2+), Zn(2+), Al(3+) and 10 mM Tris. The K(m) value of maltose conversion was 290.7 mM.

scholarly journals Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of Staphylococcus aureusBacteriophage 187

1999 ◽  
Vol 181 (15) ◽  
pp. 4452-4460 ◽  
Author(s):  
Martin J. Loessner ◽  
Susanne Gaeng ◽  
Siegfried Scherer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
De Novo ◽  
Atp Synthesis ◽  
Large Cell ◽  
Lytic Enzyme ◽  
Reading Frame ◽  
C Terminus

ABSTRACT We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to anN-acetylmuramoyl-l-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by λgt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.

scholarly journals Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants

Molecules ◽  
2020 ◽  
Vol 25 (4) ◽  
pp. 1005
Author(s):  
Yi-Ting Fang ◽  
Si-Yu Li ◽  
Nien-Jen Hu ◽  
Jie Yang ◽  
Jyung-Hurng Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Fusion Protein ◽  
Md Simulation ◽  
Cleavage Reaction ◽  
Chitin Binding Domain ◽  
First Order ◽  
C Terminus ◽  
Efficient Performance ◽  
Order Rate Equation

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

scholarly journals The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16

2017 ◽  
Vol 28 (18) ◽  
pp. 2420-2433 ◽  
Author(s):  
Yuqing Hou ◽  
George B. Witman
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Intraflagellar Transport ◽  
Critical Importance ◽  
C Terminus ◽  
N Terminus ◽  
Dynein Arms

Cilia are assembled via intraflagellar transport (IFT). The IFT machinery is composed of motors and multisubunit particles, termed IFT-A and IFT-B, that carry cargo into the cilium. Knowledge of how the IFT subunits interact with their cargo is of critical importance for understanding how the unique ciliary domain is established. We previously reported a Chlamydomonas mutant, ift46-1, that fails to express the IFT-B protein IFT46, has greatly reduced levels of other IFT-B proteins, and assembles only very short flagella. A spontaneous suppression of ift46-1 restored IFT-B levels and enabled growth of longer flagella, but the flagella lacked outer dynein arms. Here we show that the suppression is due to insertion of the transposon MRC1 into the ift46-1 allele, causing the expression of a fusion protein including the IFT46 C-terminal 240 amino acids. The IFT46 C-terminus can assemble into and stabilize IFT-B but does not support transport of outer arm dynein into flagella. ODA16, a cargo adaptor specific for outer arm dynein, also fails to be imported into the flagella in the absence of the IFT46 N-terminus. We conclude that the IFT46 N-terminus, ODA16, and outer arm dynein interact for IFT of the latter.

scholarly journals Monoclonal antibodies specific for the C-terminus of the laminin B2 subunit. Evidence for glycosylation differences between murine and human laminin

1991 ◽  
Vol 277 (3) ◽  
pp. 593-596
Author(s):  
J C Brown ◽  
J H Spragg ◽  
P W Taylor
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Fusion Protein ◽  
Adult Mouse ◽  
Yolk Sac ◽  
Specific Difference ◽  
C Terminus ◽  
Mouse Tissues ◽  
Species Specific ◽  
Beta Galactosidase

We have raised a panel of monoclonal antibodies against a beta-galactosidase fusion protein (XLB2.1) containing the C-terminal 153 amino acids of the murine laminin B2 subunit. Five of the nine antibodies characterized recognize human placental laminin as well as murine Engelbreth-Holm-Swarm (EHS)-tumour laminin. Only two of the antibodies recognize both rat parietal-yolk-sac laminin and murine EHS-tumour laminin. Two antibodies recognize an epitope on the human laminin B2 subunit which is masked by N-linked oligosaccharide in murine EHS-tumour laminin. These antibodies also fail to bind to laminin from adult-mouse tissues. These results demonstrate a species-specific difference in the glycosylation of the laminin B2 subunit.

Characterization of three terpenoid glycosyltransferase genes in ‘Valencia’ sweet orange (Citrus sinensis L. Osbeck)

Genome ◽  
2010 ◽  
Vol 53 (10) ◽  
pp. 816-823 ◽  
Author(s):  
Jing Fan ◽  
Chunxian Chen ◽  
Qibin Yu ◽  
Zheng-Guo Li ◽  
Frederick G. Gmitter
Keyword(s):  
Amino Acids ◽  
Citrus Sinensis ◽  
Southern Blotting ◽  
Sweet Orange ◽  
Secondary Product ◽  
Reading Frame ◽  
C Terminus ◽  
Group D

Three putative terpenoid UDP-glycosyltransferase (UGT) genes, designated CsUGT1, CsUGT2, and CsUGT3, were isolated and characterized in ‘Valencia’ sweet orange ( Citrus sinensis L. Osbeck). CsUGT1 consisted of 1493 nucleotides with an open reading frame encoding 492 amino acids, CsUGT2 consisted of 1727 nucleotides encoding 504 amino acids, and CsUGT3 consisted of 1705 nucleotides encoding 468 amino acids. CsUGT3 had a 145 bp intron at 730–874, whereas CsUGT1 and CsUGT2 had none. The three deduced glycosyltransferase proteins had a highly conserved plant secondary product glycosyltransferase motif in the C terminus. Phylogenetic analysis showed that CsUGT1 and CsUGT3 were classified into group L of glycosyltransferase family 1, and CsUGT2 was classified into group D. Through Southern blotting analysis, CsUGT1 was found to have two copies in the sweet orange genome, whereas CsUGT2 and CsUGT3 had at least seven and nine copies, respectively. CsUGT1, CsUGT2, and CsUGT3 were constitutively expressed in leaf, flower, and fruit tissues. The results facilitate further investigation of the function of terpenoid glycosyltransferases in citrus and the biosynthesis of terpenoid glycosides in vitro.

scholarly journals Purification and Characterization of Arginine Carboxypeptidase Produced by Porphyromonas gingivalis

2002 ◽  
Vol 70 (4) ◽  
pp. 1807-1815 ◽  
Author(s):  
Kaname Masuda ◽  
Masami Yoshioka ◽  
Daisuke Hinode ◽  
Ryo Nakamura
Keyword(s):  
Amino Acids ◽  
Porphyromonas Gingivalis ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Reading Frame ◽  
Active Peptides ◽  
C Terminus ◽  
Arginine Aminopeptidase

ABSTRACT Arginine carboxypeptidase was isolated from the cytoplasm of Porphyromonas gingivalis 381 and purified by DEAE-Sephacel column chromatography, followed by high-performance liquid chromatography on DEAE-5PW and TSK G2000SWXL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of three major bands at 42, 33, and 32 kDa with identical N-terminal sequences. By Western blotting analysis and immunoelectron microscopy, the arginine carboxypeptidase was found to be widely distributed in the cytoplasm and on the surface of the outer membrane. The open reading frame corresponding to the N-terminal amino acids of the arginine carboxypeptidase was detected by a search of the sequence of the P. gingivalis W83 genome. This sequence showed homology with mammalian carboxypeptidases (M, N, and E/H) and included a zinc-binding region signature, suggesting that the enzyme is a member of the zinc carboxypeptidase family. The purified enzyme was inhibited by EGTA, o-phenanthroline, dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and some metal ions, such as Cu2+, Zn2+, and Cd2+. On the other hand, Co2+ activated the enzyme. The enzyme released arginine and/or lysine from biologically active peptides containing these amino acids at the C terminus but did not cleave substrates when proline was present at the penultimate position. These results indicate that the arginine carboxypeptidase produced by P. gingivalis is an exo type of metallocarboxypeptidase. This enzyme may function to release arginine in collaboration with an arginine aminopeptidase, e.g., Arg-gingipain, to obtain specific amino acids from host tissues during the growth of P. gingivalis.

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