Human Cystathionine beta-Synthase Variants and Methods of Production Thereof

A method for producing truncated form of human cystathionine β-synthase protein having no more than 2 non-human, non-CBS amino acid residues at the N-terminus, said method comprising:

• a) transfecting a recombinant host cell with a recombinant nucleic acid molecule comprising a first nucleic acid sequence encoding a human cystathionine β-synthase or homologue thereof spanning from a starting position of amino acid residue 1 or one of amino acid residues from 66-84 of SEQ ID NO:2 to an ending position of one of amino acid residues from 382-532 or 543-550 of SEQ ID NO:2, and having human cystathionine β-synthase biological activity, wherein the recombinant nucleic acid molecule comprises a recombinant expression vector operatively linked to said first nucleic acid sequence, said expression vector comprising:
• i) a second nucleic acid sequence encoding a fusion segment which is linked to the N-terminus of said first nucleic acid sequence by a linker region that will produce a human cystathionine β-synthase fusion protein comprising said fusion segment when said recombinant nucleic acid molecule is expressed;
• and
• ii) a rhinovirus 3C protease recognition sequence within said linker region;
• wherein the 5′ nucleotide of the nucleic acid sequence encoding the N-terminal amino acid residue of the human cystathionine β-synthase or homologue thereof is contiguous with the 3′ nucleotide of the nucleic acid sequence encoding the two amino acid residues that occur immediately C-terminal to the rhinovirus 3C protease recognition site, such that a human cystathionine β-synthase expressed by said recombinant nucleic acid molecule will contain at its N-terminus the two C-terminal amino acid residues of the rhinovirus 3C protease recognition site;
• b) culturing the transfected host cell from (a) under conditions effective to produce said recombinant human cystathionine β-synthase fusion protein;
• and
• c) contacting the recombinant human cystathionine β-synthase fusion protein with a rhinovirus 3C protease to cleave the fusion segment from the recombinant human cystathionine β-synthase to produce the truncated form of human cystathionine β-synthase protein having no more than 2 non-human, non-CBS amino acid residues at the N-terminus.