DD217823A1 - PROCESS FOR OBTAINING ERYTHROGENIC TOXIN TYPE C - Google Patents

Abstract

The process for the recovery of erythrogenic toxin type C pursues the task to obtain from culture filtrates of b-haemolysierender streptococci formed only in very small amounts erythrogenic toxin type C. The object is achieved according to the invention by binding the toxin to acid-activated silica gel at high ion concentrations and low p H values or to neutral silica gel at neutral p H values and 10-20% strength ammonium sulfate salt from the streptococci culture filtrates and separating them mechanically from the streptococci culture filtrate. The toxin is eluted from the silica gel with ethanol buffers of high p H values. By precipitation with ammonium sulfate, the crude toxin is recovered. After dissolution in water and treatment with freshly prepared calcium phosphate, the fine purification is carried out by stepwise chromatography on carboxymethyl ion exchangers. The adsorption occurs at low ion strength at low acid p H values, the toxin is eluted at low alkaline p H values and low ion strength.

Description

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  Title of the invention Process for obtaining type C erythrogenic toxin

Field of application of the invention

The invention relates to a method for the recovery of erythrogenic toxin type C in high purity. Exythrogenic toxin type C is formed in addition to the erythrogenic toxins type A and B of group A streptococci (Streptococcus pyogenes). These three types of toxins are called scarlet toxins. They are responsible for the skin redness of scarlet fever. All three soharlachtoxin types are antigenic. The scarlatina prophylaxis or therapy was therefore carried out by active immunization or passive serum therapy (H. Schmidt, Grundlagen der spec. Therapy and prophylaxis of bacterial infectious diseases, Berlin, B40, pp. 1027-1G38) with partially purified preparations, so that side effects occurred (Topley and Wielson, Principles of Bacteriology and Immunology, GSWilson and AAMiles, Edward Arnold Ltd. London 1955, Vol 2 pp. 1669-1672).

Characteristic of the known technical solutions

The known purification protocol for the erythrogenic toxin type C is based on the precipitation of streptococci culture filtrate with four volumes of ethanol, the solubility at pH 4.0 being exploited by dissolving in acidic buffers (Kim and DWWatson, A purified group A streptococcal exotoxin, Physicochemical and biological properties including the enhancement of susceptibility to endotoxin lethal shock, J. Exp. Med 131, 1970, 611-628). After subsequent treatment with hyaluronidase to remove the co-precipitated hyaluronic acid and a preparatory

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In the case of isoelectric focusing (P.M. Schlievert, IC.IiUBettin UBd .0.7 /. Watson, Purification and characterization of group A streptOGOCoal pyrogenic exotoxin Type.C, Infect. Immunity 16, 1977, 673-679) pure type C toxin was obtained. The first concentration step in the form of alcohol precipitation used in this purification is technically only usable for "limited amounts of Streptokok ™ kenkultur filtrate (high alcohol consumption, enlargement of the volume by a factor of 4 to 5)." At the same time, high-viscosity hyaluronic acid is precipitated, which makes every subsequent purification step very difficult. The preparative isoelectric focusing as a cleaning method is not technically applicable due to the high economic outlay. In patent V / P. 1572. 43 of D.Gerlach and W.Kohler a method for the adsorption of erythrogenic toxin from Streptokokkenkulturfiltraten is described in which Acid streptococci culture filtrates at low ionic strengths or from neutral streptococcus culture at high ionic strengths with acid-activated magnesium silicate adsorbs the toxin and purifies the toxin on GM-Sepharose chromate ograf ie in acid pH at pH 5.2, according to this Proceed n only erythrogenic toxin type A can be adsorbed and purified «

Object of the invention.

The invention aims to obtain erythrogenic toxin type C.

Presentation of the invention of the invention

The invention has for its object to describe a method with which highly purified erythrogenic toxin type C can be obtained.

It has been found that from a fermented Streptokokkeke.n culture after killing germs erythrogenic toxin type C, which can be adsorbed from neutral as well as acid Streptokokkenkulturfiltraten neither at low nor at high ionic strengths with acid-activateda Mägnesiumsilikat, but is adsorbed on 'silica gel, if at low pH and D

At the same time high ionic strengths with acid-activated silica gel or at neutral pH values and 10 to 20% neutral salt concentration with neutral silica gel Streptokokkenkulturfiltrate be extracted. From the mechanically separated Adr '.s or bat, the toxin can be eluted with high pH buffers containing 5 to 10 water-miscible organic solvents. The toxin is precipitated by neutral salt and freed of foreign proteins by precipitation by stirring calcium chloride and disodium hydrogen phosphate under neutral pH · ^ · precipitated conditions Galciumphosphat and the fine purification by stepwise fractionation of Carboxyraethylionenaustauschern, preferably carboxymethylsepharose, carried out, surprisingly erythrogenic toxin type C at low ionic strengths and weak alkaline buffers eluted from the exchanger. Thus, a highly purified toxin type C is reproducibly obtained in simple and few Sohritten.

embodiments

The method will be explained in more detail by embodiments.

example 1

50 liters of streptococcus culture with 0.75 to 1 mg toxin type C per liter are brought to the end of fermentation by addition of concentrated hydrochloric acid to pH 3.5 and stirred to kill the streptococci for one hour. Subsequently, the pH is H ^r ert to pH 4.0 by addition of sodium hydroxide solution and

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centrifuged the streptococci. 3 kg of sodium chloride are dissolved in the acidic streptococcus culture filtrate and 3 kg of acid-activated silica gel (preferably silica gel B, grade 1, VEB Chemiewerk Bad Köstritz) are stirred in and allowed to settle after two hours. The supernatant is poured off and the silica gel is washed with 0.02M acetate buffer pH 5.0 containing 1M sodium chloride. With five liters of l% sodium carbonate solution containing 500 ml of ethanol, the toxin is eluted from the silica gel. 30 to 35 mg of crude toxin are obtained. For further cleaning will be

to riutuuxinlosung 2.5 kg of ammonium sulfate added and after. Leave on over TüTaoht at 4 ⁰ C left. The precipitated precipitate is separated off and the remaining supernatant is extracted with 50 g of neutral silica gel (preferably silica gel B). The bound toxin is eluted with 100 ml of 100% sodium carbonate solution and the solution is neutralized with 1N hydrochloric acid and then combined with the precipitate dissolved in 150 ml of water To this solution is added 2 g of calcium chloride (anhydrous) and stirred at a controlled pH of 7.0 to 7.6 by addition of 4 g of disodium hydrogen phosphate (dissolved in 20 ml of water) The precipitated phosphate gel is separated off from the clear solution of toxin (260 ml) with ammonium sulfate (80 $ saturation) The precipitated crude material is dissolved in water and dialyzed extensively against 0.005 M phosphate buffer pH 6.5 The precipitate which precipitates during dialysis is separated off and the clear solution is separated on a carboxymethyl ion exchange column, preferably CM-Sepharose 6 B, which had been equilibrated with the same buffer by washing with 0.005 M phosphate buffer pH 6.5 to absorbance 0 (280 nm), the g removed most of the impurities. The mixture is then eluted with a 0.007 M phosphate buffer pH 7.0 until the extinction 0 (280 nm) is reached. The toxin can be eluted clean with a buffer buffer of 0.008M pH 7.5 after changing the buffer. 30 mg of crude toxin gives 10 to 12 mg of pure product.

Example 2. ,

50 liters of streptococcus culture are brought to pH 3.5 by adding concentrated hydrochloric acid and stirred for one hour to kill the streptococci. Thereafter, the pH is adjusted to 7.0 by the addition of sodium hydroxide solution and the streptococci are centrifuged at this pH. The streptococci culture filtrate 6 kg ammonium sulfate are added and dissolved. Subsequently, 6 kg of neutral silica gel (preferably silica gel B, grade 1, VEB Chemiewerk Bad Köstrltz) stirred and stirred for two hours. Then let the silica gel

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sit, pour off the supernatant, wash the silica gel with 20% ammonium sulfate solution and elute the toxin with 10 liters of 1% sodium carbonate solution (pH 9.5). 20 to 25 mg of crude toxin are obtained. Further purification is carried out as described in Example 1.

The advantages of the method are the following: it is possible in a simple manner a concentration of toxin type C möglioh, it can be worked on an industrial scale, the concentration allows a pre-cleaning and the separation of the interfering high-viscosity hyaluronic acid. The obtained toxin type C is free from impurities in isoelectric focusing as well as in sodium dodecylsulfate electrophoresis.

Claims (1)

invention claim Process for the recovery of erythrocyte toxin type C from streptococcal culture filtrates, characterized in that for a fermented streptococcal culture after killing of the germs under appropriate conditions adsorption to a solid carrier, preferably silica gel, is carried out by with acid-activated silica gel "at low pH values ^v and at the same time high ionic strengths or - with neutral egg-gel "at neutral pH-values and 10 ~ 20 $ neutral salt concentration is worked and freed after desorption of the toxin Rohtoxln obtained from foreign proteins and purified by chromatography.