WO1999050397A2

WO1999050397A2 - Immobilized nuclease from serratia marcescens

Info

Publication number: WO1999050397A2
Application number: PCT/EP1999/001872
Authority: WO
Inventors: Ingo Franke; Alfred Pingoud; Wolfgang Wende; Gregor Meiss
Original assignee: Merck Patent Gmbh
Priority date: 1998-03-31
Filing date: 1999-03-22
Publication date: 1999-10-07
Also published as: WO1999050397A3; JP2002509719A; EP1070119A2

Abstract

Enzymatically active variants of nuclease from Serratia marcescens (nuclease [S.m.]) are disclosed, which do not dissociate into subunits. These variants of nuclease [S.m.] are obtainable by amino acid substitutions in positions which form the contact area between the subunits of native nuclease [S.m.]. These variants are especially suitable for immobilizing nuclease [S.m] onto base supports.

Description

Immobilized Nuclease from Serratia marcescens

The present invention relates to improvements related to immobilizing nuclease from Serratia marcescens (nuclease [S.m.]) and to active enzyme variants of nuclease [S.m.], which are not prone to dissociate into their subunits.

Nuclease [S.m.] commercialized under the tradename BENZONASE^® acts as endonuclease for both DNA and RNA. Because its enzymatic action yields oligonucleotides which are considered as biologically inactive, this enzyme is used for digesting genetic material in biological products, used e.g. for pharmaceutical preparations. In order to separate the enzyme easily from the reaction mixture, it has been proposed to use the enzyme immobilized on a carrier. However, due to the fact that active nuclease [S.m.] is formed by two identical subunits, such subunits bleed from preparations of immobilized nuclease [S.m.]. Consequently a constant loss of activity is observed when such preparations of immobilized nuclease [S.m.] are stored.

Structural data on nuclease [S.m.] are disclosed by M.D. Miller & K.L.

Krause (1996) Protein Science 5, 24 - 33. These authors describe also a monomeric variant (S179C) of the wild type nuclease [S.m.]. However this variant displays only 0.1 % residual enzymatic activity compared to the wild type.

The problem to be solved by the present invention is to provide variants of nuclease [S.m.], which cannot dissociate into subunits and which are active against nucleic acids. Such enzyme variants should be useful to be immobilized on a carrier by procedures known per se. Experiments have shown that there is at least one area within the structure of the monomer units of nuclease [S.m.] which can be modified in a way that the resulting enzyme is active and does not dissociate into subunits. This area has been shown to be the portion of the surface where both subunits are close to each other. These modifications are the key points of the present invention.

Object of the invention are variants of nuclease [S.m.] which can be optained by amino acid substitutions in positions which form the contact area between the subunits of native nuclease [S.m.]; these variants do not dissociate into subunits. Especially preferred are variants which carry at least one amino acid substitution in a position selected from: Arg 136, Asp 138, Ser 140, Asn 178, Pro 180, Ala 181 , Val 182, Asn 183, His 184, Tyr 185, Asp 225, Lys 233, Val 236, Glu 239, Leu 240, and Asn 245.

Object of the invention are immobilized nuclease [S.m.] obtainable by immobilizing one of said enzyme variants, which do not dissociate into subunits, onto a base support.

Figure 1 part A shows the tertary structure of dimeric nuclease [S.m.]; a detailed presentation is shown in part B. Figure 2 shows the position of the links formed from Cys 140; experimental details are given in example 6. Figure 3 shows the effects of varying length of the linker; experimental details are given in example 6.

SEQ.ID.NO: 1 shows the DNA sequence of the (His)₆-Gly-Ser derivative of the enzyme variant H184R; the corresponding protein sequence (SEQ.ID.NO: 2) is presented as to start at the following Asp residue in order to keep the numbering of amino acid residues the same as in the wild-type. In SEQ.ID.NO: 3 the wild type sequence of nuclease [S.m.] is presented along with the most preferred amino acid substitutions and along with the variant S179C. The (His)₆-Gly-Ser portion is presented in this sequence as an additional feature.

The selection of amino acid substitutions to produce monomeric nuclease

[S.m.] variants as well as the other variants mentioned were based on the coordinates of the crystal structure as published by M.D. Miller et al. (1994) Nature Struct.Biol. 1 461 - 468 (PDB entry ISMN). The in vitro muta- genesis which was used to obtain the variant nucleases [S.m.] S140C, S179C, H184N, H184T, and H184R was carried out using the 2-PCR method essentially as described by P. Friedhoff et al. (1994) Prot. Express. Purif. 5, 37 - 43, and by W. Ito et al. (1991 ) Gene 102, 67 - 70. The variant H184A was generated applying the inverse PCR strategy essentially as described by E. Blum et al. (1994) J.Biochem.Biophys.Meth. 29, 113 - 121. The mutations were verified by sequencing using standard procedures (ABI 373A DNA-Sequencer (Applied Biosystems) with ABI PRISM™ reaction kit and AmpliTaq™ DNA polymerase FS). The wild type nuclease [S.m.] and the respective variant enzymes were produced as His₆GlySer- tagged proteins in E. coli and purified as described by P. Friedhoff et al. (1994) Nucleic Acids Res. 22, 3280 - 3287, and by P. Friedhoff et al.

(1996) Nucleic Acids Res. 24, 2632 - 2639.

Nuclease activities of the wild-type enzyme (wt) and of the different variants were determined essentially as described by P. Friedhoff et al. (1996) Eur. J. Biochem. 24_1, 572 - 580 (hyperchromicity assay; high- molecular-weight DNA from herring sperm as substrate). Specific activities and kinetic parameters are summarized in Table 2 below.

The dimer interface of the Serratia nuclease contains a large surface area of complementary charge and shape. Together the two subunits form a single globular entity. His 184 is in a suitable distance to Pro 180, Ala 181 , Pro 182, and Asn 183 of the second subunit to form protein-protein contacts (see Figure 1 ; Table 1 ). His 184 was chosen as a prime candidate to disrupt the steric and electrostatic complementarity in the interface region as substitution of this residue promised to have a large impact on the integrity of the dimer interface. Residues were introduced that either cannot form the specific contacts to the second subunit or in addition are large and bulky and introduce additional charge. The replacement of His 184 with Ala, Asn, Thr and Arg resulted in proteins of good solubility. Solubility of the protein can further be improved by substituting one or several amino acid residues by more hydrophilic ones, e.g. by Ser; examples of suitable positions are: Arg 136, Asp 138, Asn 178, Pro 180, Ala 181 , Val 182, Asn 183, Tyr 185, Asp 225, Lys 233, Val 236, Glu 239, Leu 240, and Asn 245.

The positions mentioned above and the position Ser 140 are among the positions which form the contact area between the subunits of native nuclease [S.m.].

Table 1 Hydrogen bonds and nonbonded contacts of His 184 in the dimer interface of the Serratia nuclease. As the interactions originating from subunit A and subunit B are essentially the same interactions are given only for one subunit

Subunit 1 Subunit 2

Residue Atom Residue Atom

Intermoleculαr hydrogen bonds (< 3.5 A)

Pro 180 O His 184 NE2

Ala 181 O His 184 NE2

Val 182 O His 184 N Table 1 (continued)

Water-mediated hydrogen bonds (< 3.5 A)

H₂O-29 ND1 His 184 OH2

Nonbonded protein-protein contacts (< 3.5 A)

Pro 180 O His 184 CE1

Ala 181 C His 184 NE2

Ala 181 O His 184 CD2

Asn 183 O His 184 CE1

In addition, the S179C variant was produced that previously had been described as a monomeric variant of the nuclease [M.D. Miller & K.L. Krause (1996) Protein Science 5, 24 - 33]. The Ser 179 to Cys exchange is interesting because it would not be expected to directly disrupt the dimer interface although this residue is located near the interface (Figure 1 ). In contrast to the His 184 variants the variant S179C was only poorly expressed.

An additional variant produced was S140C. This variant was used for forming an obligatory dimer by linking the SH-groups of the two subunits. For this purpose a number of α,ω-bismaleimidoalkanes of different length has been prepared; the different α,ω-bismaleimidoalkanes have been used to link the SH-groups of the two subunits of S140C nuclease [S.m.]. The amino acid substitution S140C can also be used to introduce an binding group for immobilization of the monomeric His 184 variants mentioned above. Table 2

Specific activities and kinetic parameters for the cleavage of high- molecular-weight DNA by the wild-type Serratia nuclease and the monomeric variants S179C, H184N, H184T and H184R

activity Kcat M kcat KM variant [KU mg ¹] [M(nt)/M(E) s i^"1)] [μM(nt)] [μM^"1 s^"1] wt 3 7 * 10⁶ 1126 85 13

S179C 5 7 * 10³ 1 n d n d

H184R 5 8 * 10⁶ 1721 85 20

H184N 4 0 * 10⁶ 1197 106 11

H184T 5 7 * 10⁶ 1667 75 22

The activity data (Table 2) are in agreement with the structural data

Within the limit of error the monomeric His 184 variants have the same nucleolytic activity (10⁶ Kunits units mg^"1) and similar k_ca.- and K_M-values as the wild type enzyme, whereas the S179C variant shows only residual nucleolytic activity (10³ Kunits units mg^"1) probably due to changes in the structure The variants of nuclease [S m ] according to the present invention show considerable more nucleolytic activity than the variant

S179C, typically enzyme variants according to the invention show at least about 5 to 10 percent of the nucleolytic activity of the wild type enzyme

Most importantly, the monomeric His 184 variants have a very similar specific activity in cleaving DNA as the dimeπc wild type enzyme Taken together these results allow to conclude that the Serratia nuclease is not dependent on its dimeπc state for catalytic activity Hence, the catalytic centres of the dimeπc enzyme are completely independent of each other α,ω-bismaleimidoalkanes of different length have been prepared essentially as described by J.C. Cheronis et al. (1992) J. Med. Chem. 35, 1563 - 1572: ,ω-diaminoalkanes were reacted with maleic anhydride to yield N- ύ/s-n-maleamic acid; this product was further reacted with acetic anhydride/ sodium acetate to yield the respective α,ω-bismaleimidoalkane. The following α,ω-bismaleimidoalkanes were synthesized: bismaleimido- propane (BMP), bismaleimidobutane (BMB), bismaleimidopentane (BMPT), bismaleimidohexane (BMH), and bismaleimidoheptane (BMHP). These α,ω-bismaleimidoalkanes have been used to prepare obligatory dimers of nuclease [S.m.] based on the mutant enzyme S140S mentioned above.

The variants of nuclease [S.m.] as disclosed above can be immobilized on base supports by procedures known in the art; these procedures and their variants have been described in many hand books and reviews. Suitable base supports are also known in the art. Examples of base supports are particulate or beaded supports made of polysaccharide derivatives, or of organic polymers, or of derivatized silica; also suitable are non-particulate supports like porous membranes, spongeous materials, or woven or non- woven fabrics. Especially useful base supports are disclosed in EP 0 565 978, WO 96/00 077, and in WO 97/02 768.

For immobilizing the obligatory monomeric variants disclosed above procedures are preferred which are based on the additional substitution S140C. Immobilization is achieved by reacting the SH-group of the Cys 140 basically by procedures known in the art. For immobilizing the crosslinked dimer variants disclosed above homobidentale, trifunctional maleinimide linkers as they are disclosed in EP-A-0 618 192 are preferred. Linking procedures whereby the linker contains an SH-group, which reacts e.g. with an epoxy group on the base support are especially preferred. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preferred specific embodiments and examples are, therefore, to be construed as merely illustrative, and not limitative of the disclosure in any way whatsoever.

The entire disclosures of all applications, patents, and publications cited above and below, and of corresponding application EP 98 105 825.8, filed March 31 , 1998, are hereby incorporated by reference.

Examples

Example 1 : Synthesis of α,ω-bismaleimidopropane (BMP)

A solution of 0.05 mole n-propyldiamine in 100 ml anhydrous ether was added dropwise over 0.5 h to a well stirred solution of 0.1 mole maleic anhydride in 300 ml anhydrous ether at 0 °C. A white precipitate of N-bis-n- maleamic acid was formed immediately upon addition of the diamine. Stirring of the reaction mixture was continued for 1 h. The precipitate was collected by suction filtration and washed with ice-cold ether. The product was used in the next step without further purification.

To form the bismaleimides a mixture of 0.05 mole of W-b/s-n-maleamic acid of the previous step, 50 ml of acetic anhydride, and 10 g of dry sodium acetate was heated for 1 h at 100 °C. After removing most of the acetic anhydride by destination the reaction mixture was poured into 150 ml of iced water and stirred for 2 h. The crude product was isolated as a brownish precipitate by suction filtration. After drying over CaCI₂, volatile components were removed at 10 mbar and 80 °C. The purified product was isolated from the residue by sublimation at 0.1 mbar and 120-160 °C. It was collected as a white to off white powder. Analvsis: ¹H-NMR (CDCI₃): δ = 1.94 (quint, J = 7.15 Hz, 2H), 3.54 (t, J = 7.2 Hz, 4H), 6.71 (s, 4H) - CnHι₀N₂O₂ (234.21 ): calcd. C 56.41 , H 4.30, N 1 1.96; found C 56.14, H 4.16, N 11.69; yield: 38 % of theory (crude), 13 % (after sublimation).

Example 2: Synthesis of α,ω-bismaleimidobutane (BMB)

Using n-butyldiamine instead of n-propyldiamine α,ω-bismaleimidobutane (BMB) is prepared essentially as described in Example 1.

Analysis: ¹H-NMR (CDCI₃): δ = 1.59 (m, 4H), 3,54 (m, 4H), 6.99 (s, 4H) - Cι₂H₁₂N₂0₄ (248.24): calcd. C 58.06, H 4.87, N 11.29; found C 57.83, H 4.69, N 10.97; yield: 39 % of theory (crude), 12 % (after sublimation).

Example 3: Synthesis of α,ω-bismaleimidopentane (BMPT)

Using n-pentyldiamine instead of n-propyldiamine α,ω-bismaleimido- pentane (BMB) is prepared essentially as described in Example 1.

Analysis: H-NMR (CDCI₃): δ = 1.28 (m, 2H), 1.61 (m, 4H), 3.50 (t, J = 7.17 Hz, 4H), 6.69 (s, 4H); - C₁₃Hι₄N₂0₄ (262,26): calcd. C 59.54, H 5.38, N 10.68; found C 59.79, H 5.17, N 10.69; yield: 31 % of theory (crude), 9 % (after sublimation). Example 4: Synthesis of α,ω-bismaleimidohexane (BMH)

Using n-hexyldiamine instead of π-propyldiamine α,ω-bismaleimidohexane (BMH) is prepared essentially as described in Example 1.

Analysis: ¹H-NMR (CDCI₃): δ = 1.30 (m, 4H), 1.57 (t, J = 6.58, 4H), 3.50 (t, J = 7.17 Hz, 4H), 6.70 (s, 4H); - Cι₄H₁₆N₂0₄ (276.29): calcd. C 60.86, H 5.84, N 10.14; found C 60.62, H 5.69, N 9.98; yield: 31 % of theory (crude), 10 % (after sublimation).

Example 5: Synthesis of α,ω-bismaleimidoheptane (BMHP)

Using n-heptyldiamine instead of n-propyldiamine α,ω-bismaleimido- heptane (BMB) is prepared essentially as described in Example 1.

Analysis: ¹H-NMR (CDCI₃): δ = 1.57 (quint, J = 7.32 Hz, 2H), 1.28 (m, 8 H), 3.50 (m, 4H), 6.69 (s, 4H); - C₁₅H₁₈N₂0₄ (290,32): calcd. C 62.06, H 6.25, N 9.65; found C 61.90, H 6.26, N 9.56: yield: 32 % of theory (crude), 11 % (after sublimation).

Example 6: Crosslinking of the S140C Variant of Nuclease [S.m.] using α,ω-bismaleimidoalkanes of varying chain length

The water-insoluble crosslinking reagents were dissolved in dry DMSO at a concentration of 100 μM. The crosslinking reaction was carried out in 40 μl 30 mM Na-phosphate-buffer pH 7.0, 10 mM EDTA, 0.5 % DMSO (introduced by addition of the bismaleimidoalkane), 1 μM (based on the M_r of the monomer) nuclease [S.m.] and 0.5 μM crosslinking reagent. After incubation for 15 min with frequent shaking at room temperature, the protein was precipitated by adding 10 μl 100% trichloroacetic acid and incubation on ice for 10 min. Analysis of the reaction products was carried out by polyacrylamide gel electrophoresis on 10.5 % gels in the presence of sodium dodecyl sulfate. The gels were stained with Coomassie brilliant blue. The crosslinking yield was determined by densitometry of the stained gels using a video documentation system (Intas, Gόttingen, Germany).

Larger amounts of nuclease [S.m.] crosslinked with BMH were produced in a two step crosslinking process: 10 mg of the freshly prepared and dialyzed (10 mM Tris-HCl pH 8.2) S140C variant were incubated in 30 mM

Na-phosphate-buffer pH 7.0 at a concentration of 1 μM with 2 μM BMH for 1 h at ambient temperature. The reaction was stopped by cooling the mixture down to 0 °C. The protein was purified by binding to a Ni²⁺-NTA affinity column and removing the surplus BMH by washing the column with 10 mM Tris-HCl pH 8.2, 10 mM imidazol. After eluting the protein with 10 mM Tris-HCl pH 8.2, 200 mM imidazol, DTE was added to give a concentration of 1 mM. This mixture was incubated for two to three hours at ambient temperature to reduce oxidized thiol groups of Cys 140. Subsequently the protein was dialyzed against 10 mM Tris-HCl pH 8.2. This procedure yields 60 - 70 % of crosslinked dimer, repeating the crosslinking procedure with the product of the first crosslinking reaction described above results in yields of more than 95% of crosslinked dimer.

This crosslinked dimer was shown to be enzymatically active.

Figure 2 shows the structure of the dimer linked via the two S140C residues. The yield of the crosslinking reactions using linkers of different chain length is depicted in Figure 3.

Claims

1. Enzymatically active variants of nuclease from Serratia marcescens (nuclease [S.m.]), which do not dissociate into subunits obtainable by amino acid substitutions in positions which form the contact area between the subunits of native nuclease [S.m.].

2. Variant of nuclease [S.m.] according to claim 1 carrying at least one amino acid substitution in a position selected from: Arg 136, Asp 138, Ser 140, Asn 178, Pro 180, Ala 181 , Ala 181 , Val 182, Asn 183, His 184 Tyr 185, Asp 225, Lys 233, Val 236, Glu 239, Leu 240, and Asn 245.

3. Variant of nuclease [S.m.] according to claim 2 carrying an amino acid substitution selected from: H184A, H184N, H184T, and H184R.

4. Variant of nuclease [S.m.] according to claim 2 carrying the amino acid substitution S140C, whereby the Cys 140 residues in both subunits are covalently connected via a linker.

5. Immobilized nuclease [S.m.] containing an enzyme variant according to one of the claims 1 to 4.

6. Immobilized nuclease [S.m.] according to claim 5 whereby the linker is a trifunctional linker.