BETA-LACTAMASE VARIANTS

Abstract

The present invention relates to an isolated polypeptide having beta-lactamase activity and nucleic acid sequences encoding the polypeptide. The isolated polypeptide of the invention is a VIM-2 variant with improved properties such as improved protease stability, stability in intestinal medium, improved activity against one or more antibiotics, improved specific activity and/or improved production in a host cell.

Claims

1. A polypeptide having beta-lactamase activity, which comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, said polypeptide comprising: a substitution at each of positions 10 and 22; a substitution at each of positions 10 and 34; a substitution at each of positions 10 and 130; a substitution at each of positions 10, 22 and 34; a substitution at each of positions 10, 22 and 130; a substitution at each of positions 10, 34 and 130; or a substitution at each of positions 10, 22, 34 and 130, wherein the positions correspond to the positions in the sequence represented in SEQ ID NO:1, and wherein said polypeptide has an improved property with respect to stability as compared with wild-type VIM-2 of SEQ ID NO:1.

2. The polypeptide according to claim 1, which comprises at least one modification selected from the following: V10A; Q22H or Q22N; Q34R; and E130D.

3. The polypeptide according to claim 1, wherein the residue corresponding to the first residue of SEQ ID NO:1 is replaced with a methionine residue.

4. The polypeptide according to claim 1, further comprising a signal peptide at its N-terminal end.

5. The polypeptide according to claim 1, comprising a truncation at its N-terminal or C-terminal end as compared to the sequence shown in SEQ ID NO:1.

6. The polypeptide according to claim 5, which comprises: a C-terminal truncation of residues 237-240 of SEQ ID NO:1; a C-terminal truncation of residues 236-240 of SEQ ID NO:1; or a C-terminal truncation of residues 235-240 of SEQ ID NO:1.

7. The polypeptide according to claim 1, comprising or consisting of the amino acid sequence of any one of SEQ ID NO:4 to 23 or a fragment thereof having beta-lactamase activity.

8. An isolated nucleic acid sequence comprising a nucleic acid sequence encoding the polypeptide according to claim 1.

9. A nucleic acid construct comprising the nucleic acid sequence of claim 8, operably linked to one or more control sequences that direct the expression of the polypeptide in a suitable expression host.

10. A recombinant host cell, comprising the nucleic acid construct of claim 9.

11. A composition comprising the polypeptide of claim 1.

12. The composition of claim 11, which is orally administrable and is able to release the polypeptide in a desired part of the intestine, in particular in the jejunum, the ileum, the caecum or the colon.

13. A kit-of-parts comprising (a) the polypeptide of claim 1 or the composition of claim 11; and (b) a beta-lactam antibiotic which is sensitive to said polypeptide of (a) or contained in the composition of (a); for separate, sequential or simultaneous administration.

14. The polypeptide of claim 1, for use as a medicament.

15. A method for inactivating a beta-lactam antibiotic in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the polypeptide of claim 1, of the composition of claim 11, of the kit-of-parts of claim 13 or of the recombinant host cell of claim 10.

16. A method for treating a bacterial infection in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the polypeptide of claim 1, of the composition of claim 11, of the kit-of-parts of claim 13 or of the host cell of claim 10, wherein said polypeptide or said composition or said host cell is used in combination with a beta-lactam antibiotic which is sensitive to said polypeptide.

17. The polypeptide according to claim 2, wherein the residue corresponding to the first residue of SEQ ID NO:1 is replaced with a methionine residue.

18. The polypeptide according to claim 2, further comprising a signal peptide at its N-terminal end.

19. The polypeptide according to claim 3, further comprising a signal peptide at its N-terminal end.

20. The polypeptide according to claim 17, further comprising a signal peptide at its N-terminal end.

Description

LEGEND OF THE FIGURES

[0098] FIG. 1 is a graph representing the specific activity of wild-type VIM-2, VIM-2.sub.[Q22H,Q34R,E130D]DCT236 and VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 variants after 0, 60, 120 and 240 minutes in human ileal extract.

EXAMPLES

Example 1: Production and Purification of VIM-2 Variants

[0099] The VIM-2 variant was produced in Escherichia coli using either a T7 promoter-based expression system (using the pET-9 expression plasmid). Briefly, the mutant bla.sub.VIM-2 gene was cloned in the plasmid vector pET-9 using the Ndel and BamHI restriction sites, and the resulting plasmid introduced in E. coli BL21(DE3) cells. The resulting host cell was grown in the rich auto-inducing cell culture medium ZYP-5052 (Studier, F. W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41:207-234.) supplemented with ZnSO.sub.4 for 24 h. The bacterial cells and the culture supernatant were separated by centrifugation. The clarified culture supernatant was then concentrated using physical (e. g. ultrafiltration) or chemical (precipitation) methods. Alternatively, the protein could be extracted from the bacterial cells, which were resuspended in 100 to 200 ml of 20 mM Tris (pH 8.0) prior to treatment with physical (ultrasonication, French press) or chemical (lysozyme, detergents) agents to induce cell lysis. The cellular extract was clarified by centrifugation. The resulting clarified sample was then loaded on an anion exchange chromatography column. Proteins were eluted using a linear gradient of NaCl in 20 mM Tris buffer (pH 8.0) and the active fractions pooled and concentrated. The protein sample was then loaded on a second anion exchange column and eluted using a linear gradient of NaCl in 20 mM triethanolamine (pH 7.2). The resulting sample was loaded on a gel filtration column and the proteins eluted with 50 mM HEPES (pH 7.5) supplemented with 50 μM ZnSO.sub.4 and 150 mM NaCl. The purified protein was then concentrated and stored.

[0100] In particular, this production protocol was successfully used to produce the following variant VIM-2 enzyme: VIM-2.sub.[Q22H,Q34R,E130D], VIM-2.sub.[Q22H,Q34R,E130D]DCT236and VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236. Another variant that may be produced thanks to a similar method is VIM-2.sub.[V10A,Q22H,Q34R,E130D].

Example 2: Determination of Increased Stability of a Variant of VIM-2 in Intestinal Medium

[0101] To measure the stability of VIM-2 variants, the following procedure is applied: the imipenem-hydrolyzing activity of purified protein samples was determined after incubation in human ileal extract. The specific activity (Sp. Act.) for the variants was measured at different time points (0, 60, 120 and 240 min) during the incubation. The specific activity at time t was compared with the specific activity at time t=0 to assess the loss of activity of the variant in the intestinal extract. The change over time was expressed as the ratio between the initial activity (at t=0) and the activity measured later in time ((Sp. Act. Variant)t/(Sp. Act. Variant)t=0. A value lower than one indicates a loss of activity when incubated in the intestinal extract. The residual activity at different time points are compared to evaluate the greater stability in intestinal extract of some variants compared to the wild-type enzyme.

[0102] The specific activity of wild-type VIM-2 enzyme, VIM-2 variant VIM-2.sub.[Q22H,Q34R,E130D]DCT236 and VIM-2 variant VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 over time when incubated in ileal extract are presented in FIG. 1.

[0103] The loss of activity over time is also summarized in the following table:

TABLE-US-00005 Residual activity after x minutes in ileal extract 0 60 120 240 Wild-type VIM-2 100.0% 58.5% 17.8%  5.6% VIM-2.sub.[Q22H,Q34R,E130D]DCT236 100.0% 82.1% 69.0% 27.5% VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 100.0% 83.1% 62.6% 48.8%

[0104] As shown in the table or FIG. 1, the wild-type enzyme lost 94.4% of its activity after 240 minutes incubation in ileal extract. The VIM-2.sub.[Q22H,Q34R,E130D]DCT236and VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 variants lost only 72.5% and 51.2% of their activity in the same conditions, respectively.

[0105] The combination of substitutions Q22H, Q34R, E130D results in variants with dramatically improved properties in an industrial perspective. The addition of substitution V10A improves even further the stability of the enzyme in human ileal extract.

Example 3: Determination of the Specific Enzymatic and or Catalytic Activity of Given VIM-2 Variants Towards Various Beta-Lactams

[0106] For the variant enzymes produced as mentioned in example 1, it is possible to measure the specific activity and catalytic properties towards specific beta-lactam compounds such as beta-lactam antibiotics.

[0107] The variant VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 exhibits improved catalytic properties in buffer when considering imipenem as a substrate:

TABLE-US-00006 k.sub.cat K.sub.M K.sub.cat/K.sub.M Enzyme (S−.sup.1) (μM) (μM.sup.−1 s.sup.−1) VIM-2.sub.[Q22H,Q34R,E130D]DCT236 77 9 8.5 VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 130 14 9.4

[0108] The above results illustrate that the VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 exhibited a strongly improved activity against a carbapenem antibiotic which was unexpected and is of strong industrial interest.

[0109] Example 4: determination of the enzymatic activity of given VIM-2 variants towards various beta-lactams after 4 hours of incubation in intestinal medium

[0110] The enzymatic activity assessment is performed as described hereafter: after 4 hours incubation of VIM-2 variants in human ileal extract, the hydrolysis of beta-lactams was monitored spectrophotometrically at a suitable wavelength. The enzymatic activity may be expressed in nmol of beta-lactam substrate hydrolyzed by min and per mg of total protein in the sample.

[0111] In the following, all the enzymatic activities will be expressed relatively to the enzymatic activity of the wild-type enzyme measured in the same conditions.

[0112] For VIM-2.sub.[Q22H,Q34R,E130D]DCT236 and VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 variants, the measured enzymatic activities are:

TABLE-US-00007 (Residual Enzymatic activity of the enzyme/Residual Enzymatic activity of the wild-type enzyme) Enzyme for Piperacillin VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D]DCT236 51.44 VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 198.16

TABLE-US-00008 (Residual Enzymatic activity of the enzyme/Residual Enzymatic activity of the wild-type enzyme) Enzyme for Imipenem VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D]DCT236 20.94 VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 51.55

TABLE-US-00009 (Residual Enzymatic activity of the enzyme/Residual Enzymatic activity of the wild-type enzyme) Enzyme for Meropenem VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D]DCT236 10.52 VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 26.55

TABLE-US-00010 (Residual Enzymatic activity of the enzyme/Residual Enzymatic activity of the wild-type enzyme) Enzyme for Ceftriaxone VIM-2.sub.[WT] 1 VIM-2.sub.[Q22H,Q34R,E130D]DCT236 11.66 VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 18.59

[0113] The above results illustrate that the VIM-2.sub.[V10A,Q22H,Q34R,E130D]DCT236 exhibited a strongly improved activity after 4 hours of incubation in human intestinal medium against several beta-lactams of interest compared with the VIM-2.sub.[Q22H,Q34R,E130D]DCT236 mutant which activity against the same beta-lactams was highly improved compared with the wild-type enzyme.

[0114] The combination of substitutions V10A, Q22H, Q34R, E130D therefore results in a variant with dramatically improved properties in an industrial perspective.