G-CSF conjugates

Construction and Cloning of Synthetic Genes Encoding hG-CSF

[0269] The following DNA fragments were synthesized following the general procedure described by Stemmer et al. (1995), Gene 164, pp. 49-53:

[0270] Fragment 1, consisting of a Bam HI digestion site, a sequence encoding the YAP3 signal peptide (WO 98 / 32867), a sequence encoding the TA57 leader sequence (WO 98 / 32867), a sequence encoding a KEX2 protease recognition site (AAAAGA), a sequence encoding hG-CSF with its codon usage optimized for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0271] Fragment 2, consisting of a Bam HI digestion site, a sequence encoding the YAP3 signal peptide (WO 98 / 32867), a sequence encoding the TA57 leader sequence (WO 98 / 32867), a sequence encoding a histidine tag (SEQ ID NO:5), a sequence encoding a KEX2 protease recognition site (AAAAGA), a sequence encoding hG-CSF with its codon usage optimized for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0272] Fra...

Expression of hG-CSF in S. cerevisiae and E. coli

[0277] Transformation of Saccharomyces cerevisiae YNG318 (available from the American Type Culture Collection, VA, USA as ATCC 208973) with either plasmid pG-CSFcerevisiae or pHISG-CSFcerevisiae, isolation of transformants containing either of the two plasmids, and subsequent extracellular expression of hG-CSF without and with the HIS tag, respectively, was performed using standard techniques described in the literature. Transformation of E. coli BL21 (DE3) (Novagen, Cat. No. 69387-3) with pG-CSFcoli, isolation of transformants containing the plasmid and subsequent expression of hG-CSF in the supernatant and in the periplasm of the cell was performed as described in the pET System Manual (8th edition) from Novagen.

[0278] Expression of hG-CSF by S. cerevisiae and E. coli was verified by Western Blot analysis using the ImmunoPure Ultra-Sensitive ABC Rabbit IgG Staining kit (Pierce) and a polyclonal antibody against hG-CSF (Pepro Tech...

Generation of a Stable CHO-K1 G-CSF Producer

[0280] The day before transfection the CHO K1 cell line (ATCC #CCl-61) is seeded in a T-25 flask in 5 ml DMEM / F-12 medium (Gibco # 31330-038) supplemented with 10% FBS and penicillin / streptomycin. The following day (at nearly 100% confluency) the transfection is prepared: 90 μl DMEM medium without supplements is aliquoted into a 14 ml polypropylene tube (Corning). 10 μl Fugene 6 (Roche) is added directly into the medium and incubated for 5 min at room temperature. In the meantime 5 μg plasmid pG-CSFCHO is aliquoted into another 14 ml polypropylene tube. After incubation the Fugene 6 mix is added directly to the DNA solution and incubated for 15 min at room temperature. After incubation the whole volume is added drop-wise to the cell medium.

[0281] The next day the medium is exchanged with fresh medium containing 360 μg / ml hygromycin (Gibco). Every day hereafter the selection medium is renewed until the primary transfection pool has reach...