Orthogonal manner Purification of Recombinant Human Parathyroid Hormone (rhPTH) (1-34)

摘要

1. A method for the synthesis of rhPTH (1-34), comprising: i) Construction of cDNA encoding the chimeric nucleotide ORF, as specified in SEQ. ID NO.:1, by amplifying the cDNA by RT-PCR using gene-specific primers selected from SEQ. ID NO.:3, SEQ. ID NO.:4, SEQ. ID NO.:5, SEQ. ID NO.:6, SEQ. ID NO.:7 and SEQ. ID NO.:8, encoding rhPTH (1-34) chimeric fusion protein as specified in SEQ. ID NO. 2! ii) Expression of the chimeric fusion protein in presence of lactose as an inducer in the range of about 10-30% total protein! iii) purification of rhPTH (1-34), obtained with an orthogonal fashion, wherein the purification is carried out by cation exchange chromatography to a purity 98%, optionally followed by hydrophobic interaction chromatography (HIC) or RP-HPLC to a purity 99%. ! 2. The method of claim 1, wherein the expression vector containing the nucleotide ORF as indicated in SEQ. ID NO.:1, selected from the group consisting of pLMab, pRA, pET19b, wherein the vector is a pET 19b. ! 3. The method of claim 1, wherein the synthesis of a fusion nucleotide ORF, as specified in SEQ. ID NO.:1, is under the control of an inducible promoter selected from the group consisting of araBAD, trp, T7, lac, Pho and trc, preferably T7 promoter. ! 4. The method of claim 1, wherein the chimeric fusion partner consists essentially of 41 amino acid peptide of β-galactosidase (LacZ) Escherichia coli. ! 5. The method of claim 1, wherein the hybrid fusion protein as specified in SEQ. ID. No.:2, it consists of affine end (affinity handle) selected from the group consisting of a polyhistidine-tag, GST tag, protein and lactose binding staphylococcal protein, wherein the affinity tag is a polyhistidine-tag. ! 6. The method of claim 1, wherein the RP-HPLC column selected from the group consisting of C4, C8 and C18, wherein the column is a C4. ! 7.