Process for producing L-methionine &ggr;-lyase crystals

Details Process for producing L-methionine &ggr;-lyase crystals Process for producing L-methionine &ggr;-lyase crystals

1999-08-24

2002-11-05

Seidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Lyase

C435S069100, C435S252300, C435S320100, C424S094500, C536S023200

Reexamination Certificate

active

06475767

ABSTRACT:

TECHNICAL FIELD
This invention relates to a process for production of crystals of L-methionine &ggr;-lyase useful as an antitumor agent, a process for purification of L-methionine &ggr;-lyase comprising the process for production of crystals and recombinant L-methionine &ggr;-lyase crystals producable by the process for this producing system.
BACKGROUND ART
L-Methionine &ggr;-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxal phosphate as a coenzyme and catalyzes &agr;,&ggr;-elimination and &ggr;-replacement of L-methionine or its derivatives and also &agr;,&bgr;-elimination and &bgr;-replacement of S-substituted L-cysteine or its derivatives. It was reported that the enzyme was isolated and purified from
Pseudomonas putida
(Nakayama, T. et al., Anal. Biochem. 138, 421-424 (1984)). Recently, it was found that L-methionine &ggr;-lyase has an antitumor activity (WO94/11535). In the past, L-methionine &ggr;-lyase could be obtained in very small quantity from
Pseudomonas. putida.
However, recent development of recombinant DNA technology provides a possibility of its large quantity production (Inoue, H. et al., J. Biochem. 117, 1120-1125 (1995)).
Needless to say, a drug used as a pharmaceutical preparation should be pure. In the past, L-methionine &ggr;-lyase was extracted from cells of
Pseudomonas putida
and purified by a combination of ion-exchange column chromatographies (Nakayama, T. et al., Anal. Biochem. 138, 421-424 (1984), Lishko, V K et al., Protein expression and purification 4, 529-533 (1993)). However, such enzyme did not have an enough purity to be used as pharmaceutical preparations, and it was difficult to purify it on a large scale.
As a method for production of protein crystals, a method using polyethylene glycol is well known. Concerning L-methionine &ggr;-lyase, a procedure of its crystallization was reported (Esaki, N. et al., Methods in Enzymol. 143, 459-465 (1987)). The crystallization was performed by mixing L-methionine &ggr;-lyase with a potassium phosphate buffer containing polyethylene glycol and leaving the mixture at room temperature (so called vapor diffusion method). In the paper, however, L-methionine &ggr;-lyase of high purity which had previously been purified by column chromatographies was used for the crystallization. Moreover, the quantity of the crystals obtained was very small (1.6 mg).
DISCLOSURE OF INVENTION
It is difficult to produce a large quantity of crystals from a protein which has not been purified and contains impurities. Even though such crystallization succeeded, it was often impractical because the crystals still contain impurities. Therefore, a large scale crystallization at such step has hardly been attempted. This invention aims to provide a process for production of a large quantity of pure L-methionine &ggr;-lyase crystals from unpurified L-methionine &ggr;-lyase, and a process for purification of L-methionine &ggr;-lyase which comprises the process for production.
From the result of intensive studies for the above purpose, the present inventors have found out that a highly purified L-methionine &ggr;-lyase crystals can be produced in a short time by the method for production of L-methionine &ggr;-lyase crystals using polyethylene glycol, that is to say, the first step is the warming of a solution containing L-methionine &ggr;-lyase before or after addition of polyethylene glycol thereto and the second step is the addition of an inorganic salt. Thus, the present invention has been accomplished.
In this invention, L-methionine &ggr;-lyase means either or both of a natural L-methionine &ggr;-lyase produced by a microorganism such as
Pseudomonas putida
and a recombinant L-methionine &ggr;-lyase prepared by a recombinant DNA technique. From the point of view for industrial mass production, the recombinant L-methionine &ggr;-lyase is preferred. Therefore, though the recombinant L-methionine &ggr;-lyase (hereinafter referred to as “rMETase”) is used for explanations in the embodiments of the present invention, the natural L-methionine &ggr;-lyase can also been used.
A solution containing L-methionine &ggr;-lyase used in the process for producing the crystals of the invention means any of solutions which contain unpurified or purified L-methionine &ggr;-lyase. Examples of the solution include a crude enzyme solution described in the following step 1, a solution containing unpurified L-methionine &ggr;-lyase and a solution obtained after eliminating impurities by using polyethylene glycol. However, these examples do not limit the scope of this invention.
Additionally, cationic high molecular coagulants are used for eliminating nucleic acids or endotoxins as insoluble agglutinates by binding the cationic groups to anion groups of nucleic acids or endotoxins. Examples of the coagulants include polyethyleneimine or a cationic high molecular coagulant which mainly consists of chitosan (preferred is Kurimover I (Kurita Water Industries Ltd, Tokyo, Japan)). However, these examples do not limit the scope of this invention.


REFERENCES:
patent: 5120650 (1992-06-01), Visuri
patent: WO9411535 (1994-05-01), None
patent: WO 96-40284 (1996-12-01), None
patent: WO 99/07858 (1999-02-01), None
Nakayama et al.,Analytical Biochemistry,vol. 138, pp. 421-424 (1984).
Inoue et al.,J. Biochem.,vol. 117, pp. 1120-1125 (1995).
Lishko et al.,Protein Expression and Purification,vol. 4, pp. 529-533 (1993).
Esaki et al.,Methods in Enzymology,vol. 143, pp. 459-465 (1987).
Nobuyoshi Esaki et al, Methods in Enzymology (1987), vol. 143, pp. 459-465.
Hiroyuki Inoue et al, J. Biochem. (1995), vol. 117, No. 5, pp. 1120-1125.
Tan Y. et al., “Polyethylene glycol conjugation of recombinant methioninase for cancer therapy.” US National Library Of Medicine, Feb. 1998, 12(1), pp. 45-52, XP 002182531.

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