Concentration and Purification of Hepatitis B Antigen with Polyethylene Glycol and Polyelectrolyte 60, a Cross-Linked Copolymer of Isobutylene Maleic Anhydride

Rizzo, Edda de; Pandey, R.; Wallis, Craig; Melnick, Joseph L.

doi:10.1128/iai.6.3.335-338.1972

Cited by 14 publications

(2 citation statements)

References 15 publications

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“…Except, extraction effi ciency compare to the others was found to be less in potassium iodide and potassium thiocynate (0.011mg/ml and 0.050mg/ ml respectively). The same fi ndings were also reported by (Pointek et al, 2000;William et al, 1987;Friedman et al, 1987;Jih-han et al1995;DeRizzo et al 1972;Michel De Wilde et al 2001). The purity profi le on the SDS-PAGE was also confi rmed by intensity of bands after staining the gel and if their intensity was not varying much from sample to sample (results are shown in fi gure 2) the same type results have also been confi rmed by Craig et al (1987).…”

Section: Bioscience Biotechnology Research Communications Purificatiosupporting

confidence: 80%

Purifi cation of recombinant subunit vaccine by HbsAg cell lysis using various enrichments of chaotropic and non-ionic detergents

Ali¹,

Khan²,

Sahib³

et al. 2016

Biosci. Biotech. Res. Comm

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Hansunella polymorpha is extensively used as production strain for expressing recombinant proteins including recombinant Hepatitis B surface antigens (HBsAg). HBsAg is expressed in H. polymorpha and remains bounded to the membrane. Yeast cells are having very tough cell wall because of this; there is very little effect of chemicals on yeast cells for disruption. Some enzymes act on the yeast cells to break open the content, but their application is not feasible for industrial purposes. We established the cell lysis by using the mechanical disintegrater which gives more than 85% of cell lysis in one shot. This cell lysate was subjected to different types of chaotropic agents in different concentrations to selectively leach out the membrane bound HBsAg. Each chaotropic agent showed different levels of effect during extraction. The highest protein quantity was recorded with sodium chloride and least was with urea. Further, the quality of HBsAg extracted using urea was superior in comparison with other chaotropic agents. It was also found that the enrichment of HBsAg was found to be more in potassium thiocynate. In another experiment, the cells were exposed to the non-ionic detergents (triton X-100) before subjecting to the cell lysis. We found that there is strong link between the treatments of cells with non ionic detergent and leaching of the membrane bound protein. Very less protein could be extracted from the cells which are not exposed to non-ionic detergent. This was confi rmed by running several experiments keeping the entire cell lysis parameters constant, only cells were either exposed or non exposed.

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Section: Bioscience Biotechnology Research Communications Purificatiosupporting

confidence: 80%

Purifi cation of recombinant subunit vaccine by HbsAg cell lysis using various enrichments of chaotropic and non-ionic detergents

Ali¹,

Khan²,

Sahib³

et al. 2016

Biosci. Biotech. Res. Comm

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“…We report herein a modification of the polyethylene glycol (PEG) procedure of De Rizzo et al (5) for the isolation of HBsAg from chimpanzee plasma. It is shown that column chromatography on hydroxylapatite separates the isolated HBsAg-associated particles into populations differing in size distribution and isoelectric points.…”

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confidence: 99%

Heterogeneity of hepatitis B surface antigen-associated particles isolated from chimpanzee plasma

Vnek¹,

Ikram²,

Prince³

1977

Infect Immun

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Hepatitis B surface antigen (HBsAg) was purified from approximately 8 liters of pooled plasma from a carrier chimpanzee. Precipitation of HBsAg with polyethylene glycol resulted in more than 20-fold purification, with about 80% recovery of antigenic activity. The sample was separated by further purification and fractionation into three populations of HBsAg-associated particles by column chromatography on hydroxylapatite: the first contained short filaments and 22to 28-nm spheres, the second was composed of larger filaments and variable-sized spheres, and the third contained mostly 16to 22-nm spherical particles. A large volume of the polyethylene glycol precipitate passed through hydroxyl-apatite twice yielded over 650 mg of partially purified HBsAg. A pooled preparation of purified HBsAg was separated by zone-convection electrofocusing into five peaks of antigenic activity within the pH range of 4.7 to 5.7.The chimpanzee has been shown to be susceptible to infection with hepatitis B virus and may develop either a transient or chronic carrier state (1,7,13,14,16,17). Hepatitis B surface antigen (HBsAg) isolated from chimpanzee plasma closely resembles that isolated from human sources morphologically and serologically (6, 12).We report herein a modification of the polyethylene glycol (PEG) procedure of De Rizzo et al. (5) for the isolation of HBsAg from chimpanzee plasma. It is shown that column chromatography on hydroxylapatite separates the isolated HBsAg-associated particles into populations differing in size distribution and isoelectric points. MATERIALS AND METHODSSource of HBsAg. HBsAg of the adw subtype was purified from pooled plasma of a carrier chimpanzee previously inoculated with plasma from a chronic carrier chimpanzee.HBsAg was isolated by PEG precipitation. The resuspended PEG precipitate of HBsAg was submitted to fractionation and purification by column chromatography on hydroxylapatite. The methods of purification were modifications of those described previously (23).Precipitation of HBsAg by PEG 6000. In a prepurification step, 7,800 ml of pooled HBsAg-containing plasma was adjusted to pH 4.6, and a 30% solution of PEG (Union Carbide Co.) in distilled water was added to a final concentration of approximately 2%. The suspension was stored overnight at 4°C and clarified by centrifugation. The HBsAg in the supernatant was precipitated by raising the PEG concentration to 4%, and the antigen was separated by centrifugation after overnight storage of the suspension at 4°C. The sediment of HBsAg was resuspended to 500 ml in distilled water. The bulk of PEG and some accompanying contaminants were precipitated by adjusting the solution to pH 5.0. The sample was stored at 4°C overnight and clarified by centrifugation. The clear supernatant was readjusted to pH 4.6, and the HBsAg was precipitated by adding a 30% solution of PEG to a final concentration of 4%. The suspension was again stored overnight at 4°C, and the HBsAg was separated by centrifugation. Finally, the sediment of HBsAg was resuspended to 320...

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Characterization of hbeag by physicochemical and immunochemical methods

Katz

Melnick²,

Hollinger³

1980

Journal of Medical Virology

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HBeAg, which is a hepatitis B-associated antigen, was purified and characterized. The end product from ion exchange, gel filtration and immunoadsorbent columns consisted mainly of immunoglobulin G (IgG) molecules, identified by immunoelectrophoresis and polyacrylamide gel electrophoresis. At least four polypeptides with molecular weights of 55,000, 38,000, 25,000 and 20,000 daltons were detected in four different preparations of HBeAg. Two of these have molecular weights similar to the heavy and light chains of IgG (55,000 and 25,000 daltons). It is postulated that HBeAg may be a small ligand of 20,000 daltons with an affinity for IgG. This affinity, especially if it involves the light chain of IgG, could account for the 38,000 or 42,000 molecular weight polypeptides seen by autoradiography.

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Concentration and Purification of Hepatitis B Antigen with Polyethylene Glycol and Polyelectrolyte 60, a Cross-Linked Copolymer of Isobutylene Maleic Anhydride

Cited by 14 publications

References 15 publications

Purifi cation of recombinant subunit vaccine by HbsAg cell lysis using various enrichments of chaotropic and non-ionic detergents

Purifi cation of recombinant subunit vaccine by HbsAg cell lysis using various enrichments of chaotropic and non-ionic detergents

Heterogeneity of hepatitis B surface antigen-associated particles isolated from chimpanzee plasma

Characterization of hbeag by physicochemical and immunochemical methods

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