CS258940B1 - Method for Chromatographic Purification of Restriction Endonuclease Pst I - Google Patents

Abstract

The solution concerns a new method of chromatographic purification of restriction endonuclease Pst I, which uses column chromatography on phosphocellulose with a DEAE-cellulose column and on hydroxylapatite.

Description

The invention relates to a method for purifying Pst I restriction endonuclease from bacterial cells of the Providencia stuartii 164 strain.

Restriction endonucleases are endodeoxyribonucleases which, upon recognition of a specific double-stranded DNA sequence, cleave one phosphodiester bond in each strand. There are three classes of restriction endonucleases, of which the second class is most important for genetic engineering, including the Pst I restriction endonuclease. The endonucleases of this class cleave double stranded DNA directly in or in close proximity to the recognition sequence.

The purification of the restriction endonucleases from the cell extract employs in particular column chromatography methods, often requiring removal of nucleic acids which may interfere with the chromatographic steps. Nucleic acids can be removed from the cell extract by gel filtration, by passage of the cell extract through DEAE-cellulose under conditions in which the restriction endonuclease is not captured, as well as by streptomycin sulfate precipitation or polyethyleneimine precipitation. Several procedures for the purification of restriction endonuclease Pst I have been published.

Procedure using DEAE-cellulose and phosphocellulose chromatography (Smith, D. 1, Blattner, FR and Davies, J .: Isolation and partial characterization of the new restriction endonuclease from Providencia stuartii. NUCL. ACID. RES. 3, 343, 1976) an enzyme with very low stability was obtained. Gel filtration, phosphocellulose chromatography, and DEAÉ-cellulose chromatography (Brown, NL and Smith, M .: Single site mapping in X174am3 DNA replication form) were used to determine the Pst I restriction endonuclease recognition sequence. cleaved by restriction endonuclease Pst I. FEBS Lett., 65, 284, 1976).

Other techniques involved blue-Sepharose or pyran-Sepharose hematology after removal of nucleic acids (George, J. and Chirikjian JG: Biospecific Fractionation Matrices for Sequence Specific Endonuclease. NUCL. ACID. RES. 5, 2223, 1978), both of which procedures have failed to remove contaminating nucleases. The general purification procedure, also recommended for Pst I restriction endonuclease, utilizes phosphocellulose and hydroxylapatite chromatography (Greene, P.J., Heyneker, H.L., Bolivar, F., Rodriquez, R.L., Betlach,

M.C., Covarrubias, A.A., Backman, K. ', Russel, D.J., Tait, R., and Boyer, H., A general method for purification: of: restriction enzymes. NUCL. ACID. RES., 5, 2373, 1978).

Since the purification process did not yield a sufficiently pure enzyme, a new process was developed which guarantees not only sufficient purity of the purified enzyme but also higher yield. The proposed procedure consists of two chromatographic steps - chromatography on phosphocellulose and hydroxylapatite, where in the first step a phosphocellulose column is preceded by a DEAE-cellulose column which captures nucleic acids and a part of proteins, including a considerable amount of non-specific endonucleases, thereby substantially increasing both steps. Preferably, the DEAE-cellulose and phosphocellulose columns can be combined so that the cell extract is applied to the phosphocellulose via DEAE-cellulose in the first purification step.

The ionic strength of the isolation phosphate buffer must be adjusted to 0.12 mol / l NaCl, since under these conditions the restriction endonuclease Pst I binds to phosphocellulose but not to DEAE-cellulose, whereas nucleic acids and a large portion of contaminating proteins are retained on DEAE-cellulose. . Combining both columns will substantially reduce the time the enzyme is free in solution, where its stability is less than when bound to the support. The stability of the Pst I restriction endonuclease during purification can also advantageously be enhanced by the presence of Triton X-100 (0.15%) in the isolation buffer.

Description of a particular embodiment.

Bacterial cells are suspended in Buffer A (10 mmol / L K 2 PO 3 -I 4 HPO 4, 0.12 mol / L NaCl, mmol / L EDTA, 7 mmol / L 2-ME, pH 7.0), added with 100 µg / ml lysozyme and 25 µg / ml PMSF and stirred for 1 hour under ice-cooling. All other operations are performed at 0-6 ° C. Cells are disrupted by sonication and after cell debris is removed by ultracentrifugation, the supernatant is briefly dialysed against buffer A containing 0.15% Triton X-100, and loaded onto coupled DEAE-cellulose and phosphocellulose columns. After washing both columns with Buffer A with 0.15% Triton X-100, the phosphocellulose enzyme is eluted with a NaCl gradient (0.12 to 0.6 M NaCl).

The active fractions were pooled, briefly dialysed against buffer B (10 mmol / l Kf · PO PO-KjHPO ^),

0.2 mol / L NaCl, 3 mmol / L EDTA, 7 mmol / L 2-ME, 0.15% Triton X-100, pH 7.0); instead of dialysis, NaCl concentration can also be adjusted by dilution. After dialysis, the enzyme is applied to a hydroxylapatite column, the column is washed with buffer B, and the enzyme is eluted with a gradient of 0.01-0.4 mol / l KH2 PO4 -K2 HPO4. The active fractions were pooled, dialyzed into storage buffer (10 mmol / l Tris-UCl, 0.2 mol / l NaCl, 0.1 mmol / l EDTA, 7 mmol / l 2-ME, 0.15% Triton X -100, 50% glycerol, pH 7.4 at 20 ° C) and stored at -20 ° C.

Used shortcuts

DEAE - diethylaminoethylEDTA - ethylenediaminetetraacetic acid

2-ME-2-mercaptoethanol

PMSF - phenylmethylsulfonylfluoride

Tris - tris- (hydroxymethyl) -aminomethane

Claims (1)

Method for chromatographic purification of restriction endonuclease Pst I, characterized in that a column chromatography on phosphocellulose is used with a DEAE-cellulose prefilter column wherein the ionic strength of the phosphate isolation buffer is adjusted to 0.12 mol / l NaCl, followed by hydroxylapatite chromatography.