CS272934B1 - Method of hind ii and hind iii enzymes purifications for genic handlings - Google Patents

Abstract

A method of purification of the restrictive endonucleases Hind II and Hind III by means of vertical liquid chromatography, whereby both activities are separated in columns of phosphocellulose or Blue-cellulose or heparin-cellulose, preferably by combination of these columns and possibly with a column of DEAE-cellulose, which provides higher effectiveness of separation than with a DEAE-cellulose column alone.

Description

(57) A method of purifying restriction endonucleases Hind II and Hind III by column liquid chromatography such that both activities are separated on phosphocellulose or blue-cellulose or heparin-cellulose columns, preferably by a combination of these columns and optionally an OEAE-cellulose column, thereby achieves a higher separation efficiency than on DEAEcellulose alone.

CS 272934 Bl

CS 272 934 Bl

The invention relates to the use of sorbents of Blue-cellulose, heparin-cellulose and phosphocellulose, optionally in combination with DEAE-cellulose in the purification of restriction endonuclease enzymes Hind II and Hind III from Haemophilus influenzae Rd.

The activities of both enzymes are found in the strain together. For their use in molecular biology, it is important to best divide both activities. The general method of enzyme purification utilizes steps such as cell disintegration, centrifugation, and column chromatography.

Only the DEAE cellulose column (eg Old, Murray, Boises, 3rd Mol. Biol. 92, p. 33, 1975) is used to separate both activities in the published works. Other purification steps serve to separate non-specific impurities, not to separate both activities. Indeed, most sorbents bind both enzymes approximately equally strongly and do not allow efficient separation. Under suitable conditions, only the Hind II enzyme and the Hind III enzyme are collected on a DEAE-cellulose column.

Hind III is captured by another column, such as phosphocellulose, but which does not remove Hind II activity residues contaminating Hind III. The enzymes are then eluted with an ionic strength gradient and transferred to a suitable storage solution. The enzyme prepared in this way is suitable for a number of applications, but in some cases where excess enzyme needs to be used, even minor contamination is a second activity of defect. The present invention solves this problem by using other new columns to divide the activities.

In addition to DEAE-cellulose, the new process uses the following sorbents:

Phosphocellulose: Both activities elute close to each other, which can be used for pretreatment but cannot be used to get rid of traces of the other activity.

Blue-cellulose: Captures most of Hind III, does not capture Hind II.

Heparin-cellulose: Captured Hind II and a small portion of Hind III, most Hind III do not capture. On elution, Hind III traces are released earlier and do not contaminate Hind II.

Isolation solutions are typically potassium phosphate solutions at a concentration of about 10 mM or Tris at a concentration of about 20 mM. They also contain conventional 1M EDTA and 7 mM 2-mercaptoethanol. The NaCl concentration is variable, with the adsorption of the enzyme on the column being in the range of 0 to 0.1 M and at the end of the gradient in the range of 0.5 to 1.0 M. The pH of the solutions is in the range of 7 to 8. These conditions can be varied. over a relatively wide range. The storage solution additionally contains 50 percent glycerol. The volume of the gradient is about 10 times the column volume and the flow rate is about half the column volume per hour.

Example 1

200 ml of cell extract was prepared from 50 g cell paste (Haemophilus influenzae Rd). Nucleic acids were removed from it by precipitation with 1.3% streptomycin sulfate and the protein fraction separated between 50 and 70 percent saturation with ammonium sulfate. This sulfate fraction was dialyzed against the isolation buffer and loaded onto a phosphocellulose column (200 mL). Enzyme activities were eluted with a NaCl gradient and the fractions with predominant Hind II and Hind III activity were pooled separately. The pooled Hind III fractions were loaded onto a DEAE-cellulose column (BOml). There captured Hind II, but Hind III did not. The run-through solution with Hind III was loaded onto a Blue-cellulose column where Hind III was collected and the last traces of Hind II were not captured. The Hind III enzyme was eluted with a NaCl gradient and transferred to a storage solution. Hind II pooled fractions were loaded onto the same DEAE-cellulose column and Hind III residues were not collected. Hind II was eluted with a NaCl gradient and loaded onto a heparin-cellulose column (40 mL). Hind III-free Hind III was eluted with a NaCl gradient and transferred to the storage solution. Both enzymes can be further purified on other columns, e.g., hydroxylapatite.

Example 2

200 ml cell extract of 50 g bacteria was loaded onto a phosphocellulose column (200 ml) and the enzyme activities were eluted with a NaCl gradient. Fractions with predominant Hind II and Hind III activity were pooled separately. Hind III pooled fractions were loaded onto a Blue-cell CS 272 934 B1 t column

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lots (60 ml) where Hind III was captured but Hind II flowed. Both the solution and the combined Hind II fractions were loaded onto a heparin-cellulose column (60 mL). Both enzymes on both columns were eluted with NaCl gradients and transferred to storage solution.

Example 3

The sulphate fraction of Example 1 was loaded onto a Blue cellulose column (100 mL) followed by a heparin cellulose column (100 mL). Hind III caught on the first column and Hind II on the second. Both enzymes on both columns were eluted with NaCl gradients and transferred to storage flow.

Example 4

The cell extract was loaded onto a Blue cellulose column (100 mL). Hind II did not get caught and was in overflowing solution. Hind III was eluted with a NaCl gradient. Both enzymes were transferred to storage solution. Both enzymes can be purified on additional columns.

The foregoing examples illustrate but do not limit the method of the invention.

Claims (1)

SUBJECT OF THE INVENTION A method for purification of restriction endonucleases Hind II and Hind III by column liquid chromatography, characterized in that both activities are separated on phosphocellulose or blue-cellulose or heparin-cellulose columns, preferably by combinations of these columns and optionally with a DEAE-cellulose column.