Engineered primate L-methioninase for therapeutic purposes
Methods and compositions relating to the engineering of an improved protein with methionine-γ-lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.
1. A nucleic acid comprising a nucleotide sequence encoding a modified human cystathionine-γ-lyase (CGL) enzyme having at least the following substitutions relative to a native human CGL amino acid sequence (see SEQ ID NO: 1), said substitutions selected from (a) E59N, S63L, L91M, R119L, K268R, T311G, E339V and I353S; (b) E59I, S63L, L91M, R119L, K268R, T311G, E339V and I353S; (c) E59N, S63L, L91M, R119A, K268R, T311G, E339V and I353S; and (d) E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S, wherein the modified human CGL enzyme has higher methionine-γ-lyase activity compared to hMGL-NLV.
2. The nucleic acid of claim 1 , wherein the nucleic acid is codon optimized for expression in bacteria, fungus, insects, or mammals.
3. An expression vector comprising the nucleic acid of claim 2 .
4. A host cell comprising the nucleic acid of claim 2 .
5. The host cell of claim 4 , wherein the host cell is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.
6. The host cell of claim 5 , wherein the bacterial cell is an E. coli strain having deletions of genes ilvA and metA.
7. The nucleic acid of claim 1 , wherein the encoded enzyme has at least the substitutions E59N, S63L, L91M, R119L, K268R, T311G, E339V and I353S.
8. The nucleic acid of claim 1 , wherein the encoded enzyme has at least the substitutions E59I, S63L, L91M, R119L, K268R, T311G, E339V and I353 S.
9. The nucleic acid of claim 1 , wherein the encoded enzyme has at least the substitutions E59N, S63L, L91M, R119A, K268R, T311G, E339V and I353S.
10. The nucleic acid of claim 1 , wherein the encoded enzyme has at least the substitutions E59I, S63L, L91M, R119A, K268R, T311G, E339V and I353S.
11. The nucleic acid of claim 1 , wherein the encoded enzyme further comprises a heterologous peptide segment.
12. The nucleic acid of claim 1 , wherein the heterologous peptide segment is an XTEN peptide, an IgG Fc, an albumin, or an albumin binding peptide.