Modified beta-lactamase and method for its preparation
The invention relates to targeted post translational modification of metallo-beta-lactamase by truncation and insertion of a dipeptide at the amino terminal end to reduce amino terminal heterogeneity in a recombinant DNA production system. A protein K-T-E-ΔBL is expressed, and modified by host proteases to E-ΔBL. Appropriate nucleotide molecules, vectors and hosts are also described. E-ΔBL is useful in a pharmaceutical composition for treating antibiotic induced adverse effects in the intestine of patients treated with beta-lactam antibiotics.
1. A truncated metallo-beta-lactamase protein having the general formula
NH 2 -E-ΔBL-COOH
wherein
E is glutamic acid, and
ΔBL is a metallo-beta-lactamase protein that has been truncated at the amino terminal end so as to leave four beta-strands before the first alpha-helix of the predicted secondary structure of said protein, wherein E-ΔBL has an amino terminal end that corresponds to amino acid residues 12-15 of SEQ ID NO:1, and the amino acid at position +13 is either threonine or alanine.
2. The metallo-beta-lactamase protein of claim 1 , which belongs to subgroup B1 of metallo-beta-lactamases.
3. The metallo-beta-lactamase protein of claim 2 , wherein the truncated beta-lactamase is obtained from Bacillus spp, and especially from B. cereus.
4. The metallo-beta-lactamase of claim 3 , wherein E-ΔBL is obtained by deleting the first eleven amino terminal amino acids of a mature metallo-beta-lactamase.
5. The metallo-beta-lactamase of claim 2 , wherein E-ΔBL is obtained by truncation of a metallo-beta-lactamase between the amino acids KN and E.
6. A pharmaceutical composition comprising the truncated modified metallo-beta-lactamase protein of claim 1 .
7. The truncated metallo-beta-lactamase of claim 1 , wherein E-ΔBL has an amino terminal end that corresponds to amino acid residues 12-19 of SEQ ID NO:1, and the amino acid at position +13 is either threonine or alanine.
8. The truncated metallo-beta-lactamase of claim 1 , wherein E-ΔBL has an amino terminal end that corresponds to amino acid residues 12-23 of SEQ ID NO:1, and the amino acid at position +13 is either threonine or alanine.
9. The metallo-beta-lactamase protein of claim 2 , wherein the sequence E-ΔBL has at least 90% identity to the amino acid sequence of sequential amino acid residues 6-221 of SEQ ID NO:3.
10. The metallo-beta-lactamase protein of claim 2 , wherein the sequence E-ΔBL has at least 95% identity to the amino acid sequence of sequential amino acid residues 6-221 of SEQ ID NO:3.
11. The metallo-beta-lactamase protein of claim 2 , wherein the sequence E-ΔBL has at least 98% identity to the amino acid sequence of sequential amino acid residues 6-221 of SEQ ID NO:3.
12. The pharmaceutical composition of claim 6 , wherein at least 90% of the metallo-beta-lactamase is present in a single form as the metallo-beta-lactamase of claim 8 .