scholarly journals Construction and Evaluation of a Novel Bifunctional N-Carbamylase–d-Hydantoinase Fusion Enzyme

2000 ◽  
Vol 66 (5) ◽  
pp. 2133-2138 ◽  
Author(s):  
Geun-Joong Kim ◽  
Dong-Eun Lee ◽  
Hak-Sung Kim
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Intact Protein ◽  
E Coli ◽  
Fusion Enzyme ◽  
N Terminus ◽  
Fusion Enzymes ◽  
Hydantoin Derivatives

ABSTRACT A fully enzymatic process employing two sequential enzymes,d-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production ofd-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the d-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli was fused withd-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of natived-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics ofd-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids [aa]) from Agrobacterim radiobacter NRRL B11291 andd-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to thed-amino acids. A related d-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.

scholarly journals Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

2008 ◽  
Vol 190 (11) ◽  
pp. 4001-4016 ◽  
Author(s):  
Wallace A. Kaserer ◽  
Xiaoxu Jiang ◽  
Qiaobin Xiao ◽  
Daniel C. Scott ◽  
Matthew Bauler ◽  
...  
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Outer Membrane Proteins ◽  
Binding Motif ◽  
Inner Membrane ◽  
E Coli ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus

ABSTRACT We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB + bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

In-fusion expression and characterization of β-xylanase and β-1,3-1,4-glucanase in Pichia pastoris

Biologia ◽  
2012 ◽  
Vol 67 (4) ◽  
Author(s):  
Jiayun Qiao ◽  
Yunhe Cao
Keyword(s):  
Bacillus Subtilis ◽  
Pichia Pastoris ◽  
Fusion Protein ◽  
Fusion Expression ◽  
Chimeric Genes ◽  
Fusion Enzyme ◽  
Fusion Enzymes ◽  
Aspergillus Sulphureus

AbstractTwo chimeric genes, XynA-Bs-Glu-1 and XynA-Bs-Glu-2, encoding Aspergillus sulphureus β-xylanase (XynA, 26 kDa) and Bacillus subtilis β-1,3-1,4-glucanase (Bs-Glu, 30 kDa), were constructed via in-fusion by different linkers and expressed successfully in Pichia pastoris. The fusion protein (50 kDa) exhibited both β-xylanase and β-1,3-1,4-glucanase activities. Compared with parental enzymes, the moiety activities were decreased in fermentation supernatants. Parental XynA and Bs-Glu were superior to corresponding moieties in each fusion enzymes because of lower Kn higher kcat. Despite some variations, common optima were generally 50°C and pH 3.4 for the XynA moiety and parent, and 40°C and pH 6.4 for the Bs-Glu counterparts. Thus, the fusion enzyme XynA-Bs-Glu-1 and XynA-Bs-Glu-2 were bifunctional.

scholarly journals Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

1994 ◽  
Vol 127 (6) ◽  
pp. 1617-1626 ◽  
Author(s):  
M Furuse ◽  
M Itoh ◽  
T Hirase ◽  
A Nagafuchi ◽  
S Yonemura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Tight Junctions ◽  
Integral Membrane Protein ◽  
Deletion Mutants ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Vitro Binding ◽  
Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

The purification from Escherichia coli of a protein relaxing superhelical DNA

1976 ◽  
Vol 54 (4) ◽  
pp. 301-306 ◽  
Author(s):  
M. G. Burrington ◽  
A. R. Morgan
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Rapid Screening ◽  
Intact Protein ◽  
Winding Number ◽  
Circular Dna ◽  
E Coli ◽  
The Individual ◽  
Negative Supercoils

The Escherichia coli omega protein was first described by Wang (Wang, J.C.: J. Mol. Biol. 55, 523–533 (1971)) as having the ability to relax supercoiled covalently-closed circular DNA by changing the topological winding number, α. We have developed a rapid assay for omega activity which has allowed us to purify the protein to homogeneity. It appears to be an αβ-ype subunit protein with a molecular weight of the intact protein of about 80 000 (determined by gel filtration) and of the individual subunits of 56 000 and 31 000 (sodium dodecyl sulfate polyacrylamide gels). We have confirmed Wang's observation that it only partly relaxes negative supercoils, and is not active on positive supercoils. Its characteristics with respect to pH, salts, temperature and chromatography are described. A method for rapid screening of E. coli for omega mutants is described.

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.

Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

2000 ◽  
Vol 20 (3) ◽  
pp. 185-197 ◽  
Author(s):  
Pavel Dibrov ◽  
Rakhilya Murtazina ◽  
James Kinsella ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Cytoplasmic Domain ◽  
Intact Protein ◽  
Affinity Column ◽  
Rich Cluster ◽  
Protein Mutation ◽  
Conserved Sequence ◽  
Carboxyl Terminal

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na+/H+exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na+/H+ exchanger.

scholarly journals A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli

1974 ◽  
Vol 139 (2) ◽  
pp. 391-398 ◽  
Author(s):  
Susan Wilkinson ◽  
Jeremy R. Knowles
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Bacillus Stearothermophilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Binding Constants ◽  
Trna Synthetase ◽  
Subunit Structure ◽  
Filtration Method ◽  
E Coli

The purification of valyl-tRNA synthetase from Bacillus stearothermophilus is described. The protein was greater than 90% homogeneous on polyacrylamide-gel electrophoresis after more than 850-fold purification. It has a molecular weight of 110000, and no evidence was found for the presence of subunit structure. The properties of the purified enzyme were compared with those of purified valyl-tRNA synthetase from Escherichia coli. The thermal stability, pH-stability and dependence of activity on the temperature and pH of the assay are reported. The two enzymes recognize and charge tRNAVal from crude tRNA of the mesophile E. coli and of the thermophile B. stearothermophilus, indiscriminately. The gel-filtration method was extended to measure the binding of tRNA to synthetase directly. Binding constants for tRNAVal to valyl-tRNA synthetase from B. stearothermophilus were determined between 5° and 60°C.

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.

The Conservation of Amino Acids in the N-Terminal Position of Ribosomal and Cytosol Proteins from Escherichia coli, Bacillus stearothermophilus, and Halobacterium cutirubrum

1975 ◽  
Vol 53 (12) ◽  
pp. 1323-1327 ◽  
Author(s):  
Alastair T. Matheson ◽  
Makoto Yaguchi ◽  
Louis P. Visentin
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Bacillus Stearothermophilus ◽  
Protein Fractions ◽  
Extreme Halophile ◽  
Terminal Position ◽  
E Coli ◽  
Terminal Residue

Alanine, methionine, and serine are the predominant N-terminal residues in the cytosol and ribosomal protein fractions from the thermophile Bacillus stearothermophilus and the extreme halophile Halobacterium cutirubrum, a similar situation to that previously found in Escherichia coli. In all three bacteria the N-terminal residues of the 30S ribosomal proteins are mainly alanine [Formula: see text] methionine > serine whereas in the 50S ribosomal proteins from E. coli and B. stearothermophilus the predominant residues are methionine > alanine > serine suggesting conservation of specific N-terminal residues in these ribosomal proteins. However, the 50S ribosomal proteins from H. cutirubrum showed serine as the major N-terminal residue.

scholarly journals Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

1974 ◽  
Vol 143 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Yarlagadda S. Prasada Rao ◽  
Joseph D. Cherayil
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Chemical Modification ◽  
Gel Filtration ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Chemical Reagents ◽  
Mild Conditions ◽  
Trna Species

35S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H2O2, NH2OH, I2, HNO2, KMnO4 and NaIO4, under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH2OH, I2 and NaIO4 reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-thiocytidine (s2C) and 2-methylthio-N6-isopentenyladenosine (ms2i6A), to various extents. CNBr, HNO2 and NaHSO3 reacted with s4U, mnm5s2U and s2C, but not with ms2i6A. KMnO4 and H2O2 were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of 35S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.

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