Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

1987 ◽  
Vol 7 (3) ◽  
pp. 231-238 ◽  
Author(s):  
P. Manjunath ◽  
M. R. Sairam ◽  
J. Uma
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Single Step ◽  
High Yield ◽  
Property A ◽  
Purification Method ◽  
Elution Pattern

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, −A2 and −A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

1990 ◽  
Vol 63 (03) ◽  
pp. 439-444 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P Walder ◽  
P W Straub
Keyword(s):  
Gel Electrophoresis ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ease Of Use ◽  
Terminal Sequence ◽  
Purification Method ◽  
Human Fibrinogen ◽  
Isolation Methods ◽  
One Step ◽  
Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Heparin Binding Proteins of Black Bengal Buck Semen and Their Correlation with Sperm Characters and Freezability

2019 ◽  
Author(s):  
M Karunakaran ◽  
Vivek C Gajare ◽  
Ajoy Mandal ◽  
Mohan Mondal ◽  
S K Das ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Heparin Binding ◽  
Sds Page ◽  
Significant Difference ◽  
Sperm Characters

This experiment was conducted to study the electrophoretic characters of heparin binding proteins (HBP) of Black Bengal buck semen and their correlation with sperm characters and cryo-survivability. Semen ejaculates (n=20/buck) were collected from nine bucks and in vitro sperm characters were evaluated at collection, after equilibration and after freeze - thawing. HBP were isolated through heparin column and discontinuous Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed to assess molecular weight. Significant difference (plessthan0.01) were observed among the bucks in sperm characters and freezability. Eight protein bands of 17 to 180 kDa in seminal plasma and 7 bands in sperm were found. 180 -136 kDa HBP of seminal plasma and 134-101 kDa HBP of sperm had showed high correlation with in vitro sperm characters. Further studies on identification of these proteins and their correlation with in vivo pregnancy are needed to find their role as marker for buck selection.

scholarly journals Rapid, high-yield production of full-length SARS-CoV-2 spike ectodomain by transient gene expression in CHO cells

2020 ◽  
Author(s):  
Matthew Stuible ◽  
Christian Gervais ◽  
Simon Lord-Dufour ◽  
Sylvie Perret ◽  
Denis L’Abbe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Transient Gene Expression ◽  
Full Length ◽  
Spike Protein ◽  
Hek293 Cells ◽  
High Yield ◽  
Diagnostic Tools ◽  
Purification Method

ABSTRACTRecombinant forms of the spike protein of SARS-CoV-2 and related viruses have proven difficult to produce with good yields in mammalian cells. Given the panoply of potential COVID-19 diagnostic tools and therapeutic candidates that require purified spike protein and its importance for ongoing SARS-CoV-2 research, we have explored new approaches for spike production and purification. Three transient gene expression methods based on PEI-mediated transfection of CHO or HEK293 cells in suspension culture in chemically-defined media were compared for rapid production of full-length SARS-CoV-2 ectodomain. A high-cell-density protocol using DXB11-derived CHOBRI/rcTA cells gave substantially better yields than the other methods. Different forms of the spike were expressed, including the wild-type SARS-CoV-2 sequence and a mutated/stabilized form (to favor expression of the full-length spike in prefusion conformation), with and without fusion to putative trimerization domains. An efficient two-step affinity purification method was also developed. Ultimately, we have been able to produce highly homogenous preparations of full-length spike, both monomeric and trimeric, with yields of 100-150 mg/L. The speed and productivity of this method support further development of CHO-based approaches for recombinant spike protein manufacturing.

Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure

1979 ◽  
Author(s):  
B Wiman
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Polyacrylamide Gel Electrophoresis ◽  
Single Step ◽  
High Ionic Strength ◽  
Chromatographic Purification ◽  
Purification Method ◽  
Activity Measurements ◽  
Chromatographic Procedure

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharose and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding ot Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.

scholarly journals Human plasma kallikrein. A rapid purification method with high yield

1981 ◽  
Vol 193 (1) ◽  
pp. 187-192 ◽  
Author(s):  
H Nagase ◽  
A J Barrett
Keyword(s):  
Human Plasma ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Yield ◽  
Gel Chromatography ◽  
Purification Method ◽  
Simple Method ◽  
Bean Trypsin Inhibitor ◽  
Rapid Purification ◽  
Sepharose 4B

A simple method for isolation of kallikrein from human plasma is described. Before activation of the enzyme with acetone, the plasma was treated with 0.2 M-methylamine at pH 8.2 to inactivate alpha 2-macroglobulin and thus prevent the irreversible binding of the active enzyme to the inhibitor. The enzyme was adsorbed on soya-bean trypsin inhibitor-Sepharose 4B and eluted with 5 mM-NaOH, pH 11.3. It was further purified by immunoadsorption of contaminating proteins, and gel chromatography on Ultrogel AcA 44. About 3 mg of kallikrein was obtained from 400 ml of plasma (35% yield). The purified enzyme was shown to be homogeneous by electrophoretic and immunological criteria. The specific activities against benzyloxycarbonylphenylalanylarginine methylcoumarylamide, prolylphenylalanylarginine methylcoumarylamide and tosylarginine methyl ester were higher than any previously reported. The purified enzyme was resolved into two forms of mol.wts. 88 000 and 86 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis without reduction. Each consisted of three chains linked by disulphide bonds, one containing the reactive serine residue (mol.wt. 36 000 or 34 000), and two additional chains (mol.wt. 28 000 and 22 000).

The interaction of lectins with the surface of differentiating erythroleukaemic cells

1979 ◽  
Vol 38 (1) ◽  
pp. 315-329
Author(s):  
R.C. Hunt ◽  
L.M. Marshall
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Erythroid Differentiation ◽  
Dimethyl Sulphoxide ◽  
Undifferentiated Cells ◽  
Globin Synthesis ◽  
Concanavalin A Receptors

Friend erythroleukaemic cells can be induced to mature along the erythroid differentiation pathway when an inducing agent such as dimethyl sulphoxide is included in the medium. In the absence of the inducing agent, the 707B line of Friend erythroleukaemic cells is highly agglutinable by the lectins concanavalin A or wheat germ agglutinin. However, 48 h after the induction of differentiation, there is a marked decrease in the agglutination of the cells in the presence of either lectin. This suggests that early in differentiation a change occurs in the cell membrane preceding the onset of globin synthesis which starts approximately 72 h after induction. The change in agglutination by concanavalin A also occurs in the presence of reagents which do not induce haemoglobin synthesis in the 707B line of Friend erythroleukaemic cells but which are able to stimulate the synthesis of this protein in other erythroleukaemic cell lines. The reduction in the agglutinability of the differentiating cells does not seem to result from a reduction in the number of concanavalin A receptors on the cells, nor does it reflect a change in the clustered distribution of concanavalin A receptors in the differentiating cells. Both the control and dimethyl sulphoxide-induced cells show a similar patchy distribution of ferritin-labelled concanavalin A when examined by electron microscopy. Polyacrylamide gel electrophoresis shows little change in the total pattern of protein synthesis by control and differentiating cells when pulse-labelled with [35S] methionine. However, use of 125I-labelled concanavalin A to stain polyacrylamide gels, on which the total proteins of control and differentiating cells had been separated, revealed a profound change in the composition of the concanavalin A-binding proteins. The control, undifferentiated cells contained eleven or more classes of concanavalin A-binding glycoproteins, many of which stained to a lesser degree as the cell density increased. After the onset of differentiation, 2 new concanavalin A-binding glycoproteins appeared within 48 h. One of these proteins has a molecular weight in excess of 180 000 while the other migrated with an apparent molecular weight of approximately 100 000. After erythroid differentiation had progressed for 120 h, these newly synthesized glycoproteins became the major concanavalin A-binding proteins of the erythroleukaemic cells.

scholarly journals Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e91380 ◽  
Author(s):  
Przemysław Płociński ◽  
Daniel Laubitz ◽  
Dominik Cysewski ◽  
Krystian Stoduś ◽  
Katarzyna Kowalska ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Single Step ◽  
Purification Method ◽  
Protein Partners

scholarly journals Effect of Multiple Mutations in the Hemoglobin- and Hemoglobin-Haptoglobin-Binding Proteins, HgpA, HgpB, and HgpC, ofHaemophilus influenzae Type b

1999 ◽  
Vol 67 (6) ◽  
pp. 2729-2739 ◽  
Author(s):  
Daniel J. Morton ◽  
Paul W. Whitby ◽  
Hongfan Jin ◽  
Zhen Ren ◽  
Terrence L. Stull
Keyword(s):  
Haemophilus Influenzae ◽  
Insertional Mutagenesis ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Triple Mutant ◽  
Gene Encoding ◽  
Purification Method ◽  
Insertional Mutation ◽  
Ofhaemophilus Influenzae ◽  
Complete Deletion

ABSTRACT Haemophilus influenzae requires heme for growth and can utilize hemoglobin and hemoglobin-haptoglobin as heme sources. We previously identified two hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA and HgpB, in H. influenzae HI689. Insertional mutation of hgpA andhgpB, either singly or together, did not abrogate the ability to utilize or bind either hemoglobin or the hemoglobin-haptoglobin complex. A hemoglobin affinity purification method was used to isolate a protein of approximately 120 kDa from thehgpA hgpB double mutant. We have cloned and sequenced the gene encoding this third hemoglobin/hemoglobin-haptoglobin binding protein and designate it hgpC. Insertional mutation ofhgpC did not affect the ability of the strain to utilize either hemoglobin or hemoglobin-haptoglobin. An hgpA hgpB hgpC triple mutant constructed by insertional mutagenesis showed a reduced ability to use the hemoglobin-haptoglobin complex but was unaltered in the ability to use hemoglobin. A second class of mutants was constructed in which the entire structural gene of each of the three proteins was deleted. The hgpA hgpB hgpCcomplete-deletion triple mutant was unable to utilize the hemoglobin-haptoglobin complex and showed a reduced ability to use hemoglobin. We have identified three hemoglobin/hemoglobin-haptoglobin-binding proteins in Haemophilus influenzae. Any one of the three proteins is sufficient to support growth with hemoglobin-haptoglobin as the heme source, and expression of at least one of the three is essential for hemoglobin-haptoglobin utilization. Although the three proteins play a role in hemoglobin utilization, an additional hemoglobin acquisition mechanism(s) exists.

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