Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1993-06-25
1994-05-10
Lilling, Herbert J.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435815, 435847, C12N 982
Patent
active
053106709
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a process for the purification of L-asparaginase.
L-asparaginase has been reported to occur in a range of bacteria, fungi, plants and mammals and its presence in Gram-negative bacteria has been well documented (Wriston & Yellin, 1973; Wriston, 1985). L-asparaginases have considerable commercial importance because some of these enzymes particularly those from Escherichia coli (asparaginase II) and Erwinia chrysanthemi are effective against acute lymphoblastic leukaemia (Wriston, 1985). The enzymes from Erwinia and E. coli show no immunological cross-reactivity and so can provide an alternative therapy for a patient who has become hypersensitive to one of these enzymes. (Cammack et al., 1982). The enzyme from Erwinia is a tetramer of relative molecular mass 140,000, with four identical subunits, and has an unusually high isoelectric point of pH 8.6.
Hitherto, L-asparaginases have been prepared on a large scale by a combination of batch and column ion-exchange chromatography. A large number of stages are required to attain the necessary purity, with batch ion-exchange chromatography being particularly labour-intensive. Current commercial practice involves the growth of a cell culture, and then a crude protein extraction employing alkali disruption and acid precipitation. The resulting extract is then subjected to adsorption to and elution from CM-cellulose. There then follows a further acid precipitation step and chromatography steps using CM and DEAE-cellulose media. One of the major problems of the system is its complexity, thus it is not readily automated and cannot be easily adapted for continuous operation.
We have now developed a new method of L-asparaginase purification resulting in a product of astonishing quality with a simplified process and the advantage of allowing for increased automation.
According to the present invention there is provided a process for producing purified L-asparaginase which comprises (a) contacting a crude extract of L-asparaginase with a solid medium having cation exchange groups so as to adsorb L-asparaginase on the support and (b) eluting adsorbed L-asparaginase from the support, characterised in that the cation exchange groups comprise sulphonate groups and the elution step (b) is carried out at a pH which is higher than the pH used in step (a) and preferably is less than 8.0.
The cation sulphonate (--SO.sub.3.sup.-), groups may be provided, for example, by groups of the formula
The nature of the matrix of the solid medium is not unduly critical, but in order to achieve a high flow rate in a continuous process a support with a high degree of porosity is preferred. Other desirable characteristics include rigidity, low non-specific binding of protein and general chemical stability. It has been found that these properties are provided by agarose or an agarose derivative.
In carrying out the process of the invention, crude dilute extract of L-asparaginase is preferably applied to the solid medium at a pH of from 4.0 to 5.5, followed by a washing step of pH 6.0. Elution of adsorbed L-asparaginase may then be effected with a suitable buffer solution of pH in the range 6.0 to 7.5. This range is preferably between 6.5 to 7.0.
The process of the invention may be advantageous applied to the purification of the L-asparaginase of E chrysanthemi. Although as mentioned above, L-asparaginases are fairly ubiquitous in prokaryotes and eukaryotes alike, that of E chrysanthemi is preferably used for this process because of its therapeutic utility. Thus the process of the invention may be employed to purify L-asparaginases extracted form cultures of E chrysanthemi or it may be employed to purify L-asparaginase from other sources. Examples include L-asparaginase having amino acid sequences related to that of the Erwinia enzyme. In order to retain a reasonably high specific activity it is desirable to retain a close sequence homology (e.g.>90%, most preferably >95%) with the wild type. The amino acid sequence of the wild type protein is see forth below.
This pro
REFERENCES:
patent: 3594282 (1971-07-01), Kawaga et al.
patent: 3597323 (1971-08-01), Roberts
patent: 3637462 (1972-01-01), Hill et al.
patent: 4729957 (1988-03-01), Lee et al.
Lilling Herbert J.
Public Health Laboratory Service Board
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