Protein a from <i>Staphylococcus aureus</i>. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins

Hjelm, Hans; Hjelm, Katarina; Sjöquist, John

doi:10.1016/0014-5793(72)80680-x

Cited by 596 publications

(180 citation statements)

References 6 publications

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“…1. IgG were prepared by Protein A-Sepharose chromatography (19). Mr 55,000 Uk (1.5 x 105 U/mg) was from the Green Cross Corporation, Osaka, Japan; MT 33,000 Uk was generously provided by Serono, Denens, Switzerland.…”

Section: Methodsmentioning

confidence: 99%

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Vassalli¹,

Dayer²,

Wohlwend³

et al. 1984

The Journal of Experimental Medicine

410

195

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The plasminogen activator (PA) produced by freshly purified human monocytes-macrophages and histiocytic, lymphoma-derived U 937 cells was analyzed by zymography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and found to migrate with an apparent Mr of 55,000, identical to that of urokinase (Uk). By immunoprecipitation with antibodies specific for the two different types of PA, the enzyme was shown to be immunologically related to urokinase, and not to tissue PA. Urokinase was secreted in the form of the inactive Mr 55,000 zymogen prourokinase , and could be converted to the active Mr 55,000 enzyme by limited proteolysis with plasmin. Conditioned media from cultures of U 937 cells and monocytes-macrophages inhibited the fibrinolytic activity of exogenously added urokinase. Using [125I]-labeled urokinase we observed the formation of an enzyme-ligand complex, which was not dissociated by boiling in SDS and migrated with an apparent Mr 40,000 daltons higher than the free enzyme; since complexed urokinase was functionally inactivated as a PA, the ligand is an inhibitor of urokinase. This inhibitor is different from fibroblast-produced protease- nexin , in that it did not interact with thrombin. These results suggest that plasminogen activation by mononuclear phagocytes can be modulated through the secretion of both (pro)enzyme and a specific inhibitor.

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Section: Methodsmentioning

confidence: 99%

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Vassalli¹,

Dayer²,

Wohlwend³

et al. 1984

The Journal of Experimental Medicine

410

195

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“…Purified CGP (100 mg/ml) was used for the generation of custom polyclonal antibodies in rabbits by Eurogentec (Seraing, Belgium). The immunoglobulin G (IgG) fraction was purified by affinity chromatography using protein A-Sepharose CL-4B (27). To purify anti-CGP-specific IgG, CGP was blotted onto a Hybond-P membrane (Amersham Biosciences).…”

Section: Vol 74 2008 Cyanophycin In Transgenic Strains Of S Cerevimentioning

confidence: 99%

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Steinle

Oppermann‐Sanio

Reichelt

et al. 2008

Appl Environ Microbiol

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Cyanophycin [multi-L-arginyl-poly(L-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactoseinducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.Cyanophycin, also referred to as multi-L-arginyl-poly(L-aspartic acid) or cyanophycin granule polypeptide (CGP), is a nonribosomally synthesized polypeptide consisting of a poly-(aspartic acid) backbone with arginine residues linked to the ␤-carboxyl group of each aspartate by the ␣-amino group (45). CGP is a polydispersed polymer; the molecular size distribution of CGP varies with the producing host strains (1,15,18,20,30,37,52). Due to its branched structure, CGP is not degradable by a wide range of proteinases (45). Biosynthesis of CGP from aspartate and arginine requires only one enzyme, cyanophycin synthetase, which is encoded by cphA (51). CGP is insoluble at neutral pH and under physiological ionic strength, but it is soluble at low (Ͼ3) or high (Ͻ9) pH. Ziegler et al. were the first to observe a water-soluble form of CGP after the heterologous expression of cphA from Desulfitobacterium hafniense strain DSM 10664 in Escherichia coli (52). A detailed study of the solubility behavior of CGP isolated from recombinant E. coli in inorganic salts has been carried out by Füser and Steinbüchel (16). It was shown that the occurrence of the soluble form was not dependent on the origin of cphA or on the host.Recently, several putative applications for CGP and its derivatives have become available, indicating there is a need for its efficient biotechnological production (36,42,43). For the production of CGP at a technical scale, cyanobacteria were shown to be unsuitable due to their low cell...

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“…Booster injections contained 800 pg of antigen and 200p1 of alum. After the last injection, serum was collected and the IgG fraction was prepared by affinity chromatography on protein A-Sepharose (CL-4B ; Pharmacia, Uppsara, Sweden) according to the method of Hjelm et al (1972). Human serum was collected from a 50-year old male patient, recently diagnosed as having chromomycosis, and living in Gabon where chromomycosis is endemic.…”

Section: Antiseramentioning

confidence: 99%

Immunochemical characterisation of antigens and growth inhibition of Fonsecaea pedrosoi by species-specific IgG

Ibrahim-Granet

Bièvre

Jendoubi

1988

Journal of Medical Microbiology

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Protein a from Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins

Cited by 596 publications

References 6 publications

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Concomitant secretion of prourokinase and of a plasminogen activator-specific inhibitor by cultured human monocytes-macrophages.

Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

Immunochemical characterisation of antigens and growth inhibition of Fonsecaea pedrosoi by species-specific IgG

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