scholarly journals Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping

2006 ◽  
Vol 393 (3) ◽  
pp. 757-766 ◽  
Author(s):  
Tun-Hsun Kuo ◽  
Shiao-Cheng Chuang ◽  
Sing-Yang Chang ◽  
Po-Huang Liang
Keyword(s):  
Recombinant Proteins ◽  
Binding Activity ◽  
Glycosylation Site ◽  
Affinity Column ◽  
Gram Negative Bacteria ◽  
The Media ◽  
Dtt Dithiothreitol ◽  
Covalently Linked ◽  
Plasma Lectin ◽  
Lps Binding

TPL (Tachypleus plasma lectin)-1 was purified by using a Sepharose column and TPL-2 was purified from an LPS–Sepharose (LPS coupled to Sepharose matrix) affinity column, as described previously [Chiou, Chen, Y.-W., Chen, S.-C., Chao and Liu (2000) J. Biol. Chem. 275, 1630–1634] and the corresponding genes were cloned [Chen, Yen, Yeh, Huang and Liu (2001) J. Biol. Chem. 276, 9631–9639]. In the present study, TPL-1 and -2 were produced in yeast, and the recombinant proteins secreted into the media were purified and characterized. The proteins show specific PGN (peptidoglycan)- and LPS-binding activity, suggesting a role in trapping Gram-positive and Gram-negative bacteria respectively in innate immunity. Using BIAcore® assays, the dissociation constant for the TPL-1–PGN complex was measured as 8×10−8 M. Replacement of Asn74, the N-glycosylation site of TPL-1, with Asp abolishes the PGN-binding affinity, whereas the unglycosylated TPL-2 N3D mutant retains LPS-binding activity. DTT (dithiothreitol) treatment to break disulphide linkages abrogates TPL-2 activity but does not interfere with TPL-1 function. Cys4 in TPL-2 may form an intermolecular disulphide bond, which is essential for activity. As a result, the TPL-2 C4S mutant is inactive and is eluted as a monomer on a non-reducing gel. TPL-2 C6S is active and forms a non-covalently linked dimer. A model describing TPL-2 binding with LPS is proposed. These two plasma lectins that have different ligand specificities can be used for the detection and discrimination of bacteria and removal of endotoxins.

scholarly journals Lipopolysaccharide (LPS) binding protein opsonizes LPS-bearing particles for recognition by a novel receptor on macrophages.

1989 ◽  
Vol 170 (4) ◽  
pp. 1231-1241 ◽  
Author(s):  
S D Wright ◽  
P S Tobias ◽  
R J Ulevitch ◽  
R A Ramos
Keyword(s):  
Acute Phase ◽  
Binding Protein ◽  
Umbilical Vein ◽  
Acute Phase Reactant ◽  
Binding Activity ◽  
Gram Negative Bacteria ◽  
Blood Monocytes ◽  
Coated Particles ◽  
Phase Reactant ◽  
Lps Binding

Lipopolysaccharide binding protein (LBP) is an acute-phase reactant that binds bacterial LPS. We show that LBP binds to the surface of live Salmonella and to LPS coated erythrocytes (ELPS), and strongly enhances the attachment of these particles to macrophages. LBP bridges LPS-coated particles to macrophages (MO) by first binding to the LPS, then binding to MO. Pretreatment of ELPS with LBP enabled binding to MO, but pretreatment of MO had no effect. Moreover, MO did not recognize erythrocytes coated with LBP unless LPS was also added, thus suggesting that interaction of LBP with LPS results in a conformational change in LBP that allows recognition by MO. Binding of LBP-coated particles appears to be mediated by a receptor found on blood monocytes and MO but not on other leukocytes or umbilical vein endothelium. The receptor is mobile in the plane of the membrane since binding activity on MO was downmodulated upon spreading of cells on surfaces coated with LBP-LPS complexes. The receptor appears to be distinct from other opsonic receptors since downmodulation of CR1, CR3, Fc gamma RI, Fc gamma RII, and Fc gamma RIII with mAbs did not affect binding of LBP-coated particles, and leukocytes from CD18-deficient patients bound LBP-coated particles normally. Coating of erythrocytes with LBP-LPS complexes strongly enhanced phagocytosis observed in the presence of suboptimal amounts of anti-erythrocyte IgG. However, binding mediated by LBP-LPS complexes alone caused neither phagocytosis of the LBP-coated erythrocytes nor initiation of an oxidative burst. The results of our studies define LBP as an opsonin. During the acute phase, LBP can be expected to bind gram-negative bacteria and bacterial fragments and promote the interaction of coated bacteria with phagocytes.

scholarly journals Impact of media components from different suppliers on enterokinase productivity in Pichia pastoris

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ján Krahulec ◽  
Martin Šafránek
Keyword(s):  
Recombinant Proteins ◽  
Fermentation Process ◽  
High Impact ◽  
Production Level ◽  
Media Component ◽  
Component Supplier ◽  
The Media ◽  
The Difference ◽  
Optimisation Procedure ◽  
The Ideal

Abstract Background The aim of this study was to provide an information about the homogeneity on the level of enterokinase productivity in P. pastoris depending on different suppliers of the media components. Results In previous studies, we performed the optimisation process for the production of enterokinase by improving the fermentation process. Enterokinase is the ideal enzyme for removing fusion partners from target recombinant proteins. In this study, we focused our optimization efforts on the sources of cultivation media components. YPD media components were chosen as variables for these experiments. Several suppliers for particular components were combined and the optimisation procedure was performed in 24-well plates. Peptone had the highest impact on enterokinase production, where the difference between the best and worst results was threefold. The least effect on the production level was recorded for yeast extract with a 1.5 fold difference. The worst combination of media components had a activity of only 0.15 U/ml and the best combination had the activity of 0.88 U/ml, i.e., a 5.87 fold difference. A substantially higher impact on the production level of enterokinase was observed during fermentation in two selected media combinations, where the difference was almost 21-fold. Conclusions Results demonstrated in the present study show that the media components from different suppliers have high impact on enterokinase productivity and also provide the hypothesis that the optimization process should be multidimensional and for achieving best results it is important to perform massive process also in terms of the particular media component supplier .

scholarly journals Biotin binding to avidin. Oligosaccharide side chain not required for ligand association

1987 ◽  
Vol 248 (1) ◽  
pp. 167-171 ◽  
Author(s):  
Y Hiller ◽  
J M Gershoni ◽  
E A Bayer ◽  
M Wilchek
Keyword(s):  
Concanavalin A ◽  
Sugar Content ◽  
Binding Activity ◽  
Side Chain ◽  
Affinity Column ◽  
Binding Properties ◽  
Commercial Preparation ◽  
Oligosaccharide Moiety ◽  
Biotin Binding

A commercially available, purified preparation of avidin was found to comprise two polypeptide bands (Mr 18,000 and Mr 15,500 respectively). Both bands bound biotin as assessed by biotin overlays of protein blots. The Mr 15,500 polypeptide was found to differ from the Mr 18,000 polypeptide only in its sugar content. When the commercial preparation was applied to a concanavalin A affinity column, the glycosylated forms were retarded as expected, and homotypic nonglycosylated avidin tetramers which failed to bind selectively to the column were collected in the effluent. The biotin-binding properties of the nonglycosylated avidin were equivalent to those obtained for the native (glycosylated) avidin molecule, indicating that the oligosaccharide moiety is not essential for the binding activity.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

scholarly journals The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

2009 ◽  
Vol 192 (2) ◽  
pp. 568-574 ◽  
Author(s):  
Takashige Kashimoto ◽  
Shunji Ueno ◽  
Takeshi Koga ◽  
Shinji Fukudome ◽  
Hayato Ehara ◽  
...  
Keyword(s):  
Aromatic Ring ◽  
Vibrio Vulnificus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Binding Activity ◽  
Gram Negative Bacteria ◽  
Ovary Cells ◽  
In The Wild ◽  
And Function ◽  
Phenylalanine Residue

ABSTRACT Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.

scholarly journals Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1540-1544 ◽  
Author(s):  
DM Monroe ◽  
DM McCord ◽  
MN Huang ◽  
KA High ◽  
RL Lundblad ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Ix ◽  
Affinity Column ◽  
Extrinsic Pathway ◽  
Control Value ◽  
Amino Terminal ◽  
Active Site Probe ◽  
Covalently Linked

Abstract Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

An integrin from oyster Crassostrea gigas mediates the phagocytosis toward Vibrio splendidus through LPS binding activity

2015 ◽  
Vol 53 (1) ◽  
pp. 253-264 ◽  
Author(s):  
Zhihao Jia ◽  
Tao Zhang ◽  
Shuai Jiang ◽  
Mengqiang Wang ◽  
Qi Cheng ◽  
...  
Keyword(s):  
Crassostrea Gigas ◽  
Binding Activity ◽  
Vibrio Splendidus ◽  
Lps Binding

scholarly journals Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins)

1984 ◽  
Vol 218 (3) ◽  
pp. 805-810 ◽  
Author(s):  
J G Haddad ◽  
M A Kowalski ◽  
J W Sanger
Keyword(s):  
Vitamin D ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Plasma Protein ◽  
Binding Protein ◽  
Binding Activity ◽  
Single Step ◽  
Affinity Column ◽  
Vitamin D Metabolites ◽  
Molar Ratios

The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein.

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