Purification of Staphylocoagulase by a Bovine Prothrombin-Sepharose 4B Column and its Physicochemical Properties

1979 ◽  
Author(s):  
H. Igarashi ◽  
T. Morita ◽  
S. Iwanaga
Keyword(s):  
Large Scale ◽  
Aureus Strain ◽  
Affinity Column ◽  
Terminal Sequence ◽  
Active Components ◽  
Simplified Method ◽  
Covalently Linked ◽  
Ultracentrifugal Analysis ◽  
Sepharose 4B ◽  
Single Symmetrical Peak

Staphylocoagulase is known to coagulate human plasma, but not bovine’s, by forming an active molecular complex with prothrombin. However, there is no enough knowledges in respect to the molecular mechanism of prothrombin activation with staphylocoasulase. A new simplified method was developed for the large-scale purification of coagulase from culture filtrates of Staphylococcus aureus. strain st-213, using Sepharose AB covalently linked with bovine prothrombin. This affinity column adsorbed strongly the coagulase, which was eluted with 1.0 M NaSCN. The yield of coagulase activity was in the range of 75 to 85X, The purified coagulase showed a single symmetrical peak by ultracentrifugal analysis (S20,w=6.47), and it gave a single precipitin line against anti-purifled staphylocoagulase serum, as revealed by the immunodiffusion test. However, the preparation was shown to contain three active components by the isoelectric foccusing method, suggesting some microheterogeneity. The molecular weight estimated by SDS-gel electrophoresis was 71,000 (major material). No cystine residue was found in the purified material and its NH2-terminal sequence was Ile-Ile-. It is of interest that staphylocoagulase interacts strongly with bovine prothrombin-Sepharose AB, whereas it does not form any active complex with the prothrombin, unlike human prothrombin.

scholarly journals Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase

1976 ◽  
Vol 153 (2) ◽  
pp. 351-361 ◽  
Author(s):  
M J Holroyde ◽  
J M E Chesher ◽  
I P Trayer ◽  
D G Walker
Keyword(s):  
Protein Binding ◽  
Association Constant ◽  
Large Scale ◽  
Competitive Inhibitor ◽  
Specific Protein ◽  
Affinity Column ◽  
Rapid Loss ◽  
Tissue Extracts ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of “used” columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand.

Construction and Functional Analysis of the Recombinant Bacteriocins Weissellicin-MBF from Weissella confusa MBF8-1

2020 ◽  
Vol 22 (1) ◽  
pp. 115-122
Author(s):  
Amarila Malik ◽  
Elita Yuliantie ◽  
Nisa Yulianti Suprahman ◽  
Theresa Linardi ◽  
Angelina Wening Widiyanti ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Large Scale ◽  
Micrococcus Luteus ◽  
Affinity Column ◽  
Gene Expressions ◽  
His Tag ◽  
Recombinant Peptides ◽  
Weak Activity ◽  
Potential Alternative ◽  
Weissella Confusa

Background: Bacteriocins (Bac1, Bac2, and Bac3) from Weissella confusa MBF8-1, weissellicin- MBF, have been reported as potential alternative substances as well as complements to the existing antibiotics against many antimicrobial-resistant pathogens. Previously, the genes encoded in the large plasmid, pWcMBF8-1, and the spermicidal activity of their synthetic peptides, originally discovered Indonesia, have been studied. Three synthetic bacteriocins peptides of this weissellicin-MBF have been reported for their potential activities, i.e. antibacterial and spermicidal. Objective: The aim of this study was to construct the recombinant Bacteriocin (r-Bac) genes, as well as to investigate the gene expressions and their functional analysis. Method: Here, the recombinant Bacteriocin (r-Bac) genes were constructed and the recombinant peptides (r-Bac1, r-Bac2, and r-Bac3) in B. subtilis DB403 cells were produced on a large scale. After purification, using the His-tag affinity column, their potential bioactivities were measured as well as their antibacterial minimum inhibitory concentrations against Leuconostoc mesenteroides and Micrococcus luteus, were determined. Results: Pure His-tag-recombinant Bac1, Bac2, and Bac3 were obtained and they could inhibit the growth of L. mesenteroides and M. luteus. Conclusion: The recombinant bacteriocin could be obtained although with weak activity in inhibiting gram-positive bacterial growth.

scholarly journals Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology

1979 ◽  
Vol 183 (3) ◽  
pp. 615-622 ◽  
Author(s):  
M A Kerr
Keyword(s):  
Sequence Homology ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Terminal Sequence ◽  
Complement Components ◽  
Factor B ◽  
Terminal Residue ◽  
Covalently Linked

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.

scholarly journals Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50

1979 ◽  
Vol 179 (2) ◽  
pp. 333-339 ◽  
Author(s):  
A Y Strongin ◽  
D I Gorodetsky ◽  
I A Kuznetsova ◽  
V V Yanonis ◽  
Z T Abramov ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Serine Proteinase ◽  
Subtilis Strain ◽  
Bacterial Strains ◽  
Terminal Sequence ◽  
Gramicidin S ◽  
Intracellular Enzymes ◽  
Sepharose 4B ◽  
Homologous Enzyme ◽  
Strict Selection

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN′!, produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

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.

scholarly journals The Importance of Interfacial Tension in Emulsification: Connecting Scaling Relations Used in Large Scale Preparation with Microfluidic Measurement Methods

2020 ◽  
Vol 4 (4) ◽  
pp. 63
Author(s):  
Karin Schroën ◽  
Jolet de Ruiter ◽  
Claire Berton-Carabin
Keyword(s):  
Interfacial Tension ◽  
Large Scale ◽  
Measurement Methods ◽  
Scaling Relations ◽  
Active Components ◽  
Preparation Methods ◽  
Large Scale Preparation ◽  
Complex Process ◽  
Scale Preparation ◽  
Multi Phase

This paper starts with short descriptions of emulsion preparation methods used at large and smaller scales. We give scaling relations as they are generally used, and focus on the central role that interfacial tension plays in these relations. The actual values of the interfacial tension are far from certain given the dynamic behavior of surface-active components, and the lack of measurement methods that can be applied to conditions as they occur during large-scale preparation. Microfluidic techniques are expected to be very instrumental in closing this gap. Reduction of interfacial tension resulting from emulsifier adsorption at the oil-water interface is a complex process that consists of various steps. We discuss them here, and present methods used to probe them. Specifically, methods based on microfluidic tools are of great interest to study short droplet formation times, and also coalescence behavior of droplets. We present the newest insights in this field, which are expected to bring interfacial tension observations to a level that is of direct relevance for the large-scale preparation of emulsions, and that of other multi-phase products.

Purification of guinea pig liver transglutaminase using a phenylalanine-Sepharose 4B affinity column

1983 ◽  
Vol 128 (1) ◽  
pp. 202-205 ◽  
Author(s):  
Paul P. Brookhart ◽  
Philip L. McMahon ◽  
Mark Takahashi
Keyword(s):  
Guinea Pig ◽  
Affinity Column ◽  
Pig Liver ◽  
Sepharose 4B ◽  
Guinea Pig Liver

scholarly journals Chemical Characterization of a Non-Covalently Linked Peptide from Plakalbumin

1969 ◽  
Vol 22 (1) ◽  
pp. 239 ◽  
Author(s):  
R Sleigh ◽  
R Hosken MB Smith ◽  
EOP Thompson
Keyword(s):  
Chemical Characterization ◽  
Protein Component ◽  
Terminal Portion ◽  
Terminal Sequence ◽  
Covalently Linked

A 33�residue peptide isolated from plakalbumin by dissociation with urea at pH 3 is shown to be derived from the a-terminal portion of ovalbumin. The yield of proline obtained on hydrazinolysis was the same as that from ovalbumin, whereas proline was not liberated from the protein component. The N�terminal sequence of this peptide was determined by the Edman degradative procedure to be Ser. V al.Ser .Glu.Glu.Phe.Arg.Ala.Asp.

scholarly journals Large-Scale Preparation of Highly Stable Recombinant Human Acidic Fibroblast Growth Factor in Escherichia coli BL21(DE3) plysS Strain

2021 ◽  
Vol 9 ◽  
Author(s):  
Bingjieu Yu ◽  
Wenzhe Sun ◽  
Zhen Huang ◽  
Gang Sun ◽  
Le Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Chloride Concentration ◽  
Mitogenic Activity ◽  
Expression Level ◽  
Diabetic Rat ◽  
Affinity Column ◽  
Separation And Purification ◽  
Time Saving ◽  
Fermentation Parameters

In this study, the optimum human aFGF gene encoding haFGF135 was cloned in pET3c and transferred to Escherichia coli BL21(DE3) plysS. To enhance the yield of fermentation and the expression level of the target protein, the fermentation parameters, including temperature, pH, dissolved oxygen, glucose concentration, ammonium chloride concentration, induction time, and inducer (IPTG) concentration, were optimized. The optimized fermentation parameters were used in large-scale fermentation (30 L). Ion-exchange and heparin-affinity column chromatography techniques were used for separation and purification of rhaFGF135 protein. HPLC, isoelectric focusing electrophoresis, and mass spectrometry were used to detect the purity, isoelectric point, and molecular weight and peptide map of rhaFGF135 protein, respectively. Mitogenic activity of rhaFGF135 protein was detected in NIH-3T3 cells and a full-thickness injury wound diabetic rat model. The production and expression level of rhaFGF135 in the 30-L scale fermentation reached 80.4 ± 2.7 g/L culture and 37.8% ± 1.8%, respectively. The RP-HPLC and SDS-PAGE purity of the final rhaFGF135 product almost reached 100%, and the final pure protein yield was 158.6 ± 6.8 mg/L culture. Finally, the cell and animal experiments showed that rhaFGF135 retained a potent mitogenic activity. The large-scale process of rhaFGF135 production reported herein is relatively stable and time-saving, and thus, it can be used as an efficient and economic strategy for the synthesis of rhaFGF135 at the industrial level.

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