Stable recombinant adenosine deaminase
A technology of adenosine deaminase and wild-type adenosine deaminase, applied in the field of stable recombinant adenosine deaminase, which can solve the problem of activity reduction
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Embodiment 1
[0193] Construction of an E. coli expression strain expressing recombinant human ADA with a Cys to Ser change at position 74 of the mature protein
[0194] The reported 363 amino acid sequence of human adenosine deaminase (GenBank NP_000013, incorporated herein by reference) was analyzed for the presence of cysteine codons. Five positions in the mature (N-terminal Met cleaved) polypeptide encode cysteines (C74, C152, C153, C168, C261). In the designed and modified gene expressing human ADA, only 1 of these 5 cysteine codons (cysteine 74, TGC) was changed to a serine codon (TCC) (which is the 75 bits). Using standard chemical synthesis of overlapping oligonucleotide fragments, the defined polypeptide sequence (see SEQ ID NO: 3) was provided to Blue Heron Corporation (Bothell, Washington, U.S.A.) for total gene synthesis of a novel gene with Codons optimized for expression in E. coli. Briefly, this sequence was optimized for bacterial expression following standard bacte...
Embodiment 2
[0199] Construction of an E. coli expression strain expressing recombinant bovine ADA with a Cys to Ser change at position 74 of the mature protein
[0200] The purified mature ADA protein derived from a bovine intestinal preparation is a 356 amino acid protein with a deletion of the N-terminal methionine and also deletion of the last 6 C-terminal residues as predicted from the cDNA sequence (GenBank NP_776312, incorporated herein by reference). The bovine ADA amino acid sequence was analyzed for the presence of cysteine codons. Five positions in the mature polypeptide encode cysteines (C74, C152, C153, C168, C261). Only 1 of these 5 cysteine positions (cysteine 74) was changed to a serine residue in the designed and modified bovine ADA synthesis gene. This is done by inserting a serine codon (TCC) at position 74 of the mature polypeptide (or position 75 of the translated product) in place of the normal cysteine codon. This gene is also codon optimized for expression...
Embodiment 3
[0205] Purified mutant rhADA protein
[0206] Purification of the mutein rhADA was performed with a 3 chromatographic protocol developed by Enzon. Bacterial fermentation of E. coli expressing rhADA protein from a synthetic gene on plasmid pET28a (Novagen) in host cell HMS174(DE3) was performed. Rifampicin (200 μg / ml) and kanamycin (30 μg / ml) were included in minimal glycerol medium supplemented with yeast extract (30 g / l), and when the inducer IPTG was added to a final concentration of 5 mM Cells were grown to OD at 28°C 600 11. After 40 hours (OD 600 ~110), cells were harvested by centrifugation and frozen at -20°C. Briefly, thawed cell plasma (50 g) was resuspended in 1800 ml of 10 mM Tris buffer [trishydroxymethylaminomethane], 1 mM DTT, pH 8.0, and incubated with Tempest Virtis (Sentry TM , Microprocessor, Boston, MA) was homogenized at 1200 RPM for 10 seconds. The suspension was passed through a stainless steel screen (Opening micrometer 250[mu], No. 60, W.S Tyler) t...
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