Extending the serum half-life of G-CSF via fusion with the domain III of human serum albumin

Abstract

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned into pPICZ α A along with the open reading frame of the α -factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed into Pichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.

Figure 1

Construction of recombinant pPIC-3DHSA-G-CSF eukaryotic expression vector. The synthesized HSA and G-CSF genes were used as templates for the amplification of 3DHSA-G10 and H10-G-CSF genes by PCR with primer 1 and primer 2, primer 3 and primer 4, respectively. The purified 3DHSA-G10 and H10-G-CSF genes were used as templates for obtaining 3DHSA-G-CSF gene by overlap PCR. Both pPICZαA plasmid and 3DHSA-G-CSF gene were digested by XhoI and ligated together by T4 DNA ligase to construct expression vector pPIC-3DHSA-G-CSF. 3DHSA-G-CSF was inserted into the sites XhoI of pPICZαA along with the open reading frame of the α-factor signal under the control of the AOX1 promoter. The KEX2 cleavage site was located between the signal peptide and the 3DHSA-G-CSF sequences.

Figure 2

Analysis of transformants that expressed 3DHSA-G-CSF by SDS-PAGE. The culture supernatants of screened transformant were resolved by 12% SDS-PAGE and stained with Coomassie Blue. M: protein marker; Lanes 1–8 correspond to different transformants of GS115-pPIC-3DHSA-G-CSF; the protein band about 42 kDa was marked 3DHSA-G-CSF on the right of SDS-PAGE.

Figure 3

Western blotting analysis using primary antibody anti-G-CSF and anti-HSA. M: prestained protein marker, (a) antibody anti-G-CSF, Lane a1: 3DHSA-G-CSF, Lane a2: G-CSF; (b) antibody anti-HSA, Lane b1: 3DHSA-G-CSF, Lane b2: HSA.

Figure 4

Purification of 3DHSA-G-CSF. The supernatants containing the target proteins were purified by Cibacron Blue F3G-A Sepharose and Butyl-Sepharose 4B column, and the final purity was detected by 12% SDS-PAGE. (a) The hydrophobic chromatograph of 3DHSA-G-CSF by Butyl-Sepharose 4B column. (b) 12% SDS-PAGE, M: protein marker, Lanes 1~3: flow through, Lanes 4~6: elute.

Figure 5

Structural analysis of purified fusion protein by peptide mass mapping and circular dichroism spectrum. (a) Peptide mass mapping of 3DHSA-G-CSF, (b) circular dichroism spectra of 3DHSA-G-CSF.

Figure 6

In vivo bioactivity of the purified 3DHSA-G-CSF. Both G-CSF and 3DHSA-G-CSF could increase the white blood cell counts in a cyclophosphamide-induced neutropenia model murine, and more potent stimulate was observed compared with CTX (P < 0.01). G-CSF versus 3DHSA-G-CSF not significant (ns) (P > 0.05). Values are expressed as mean ± SD among six samples from one experiment. Data are representative of three independent experiments with similar result.

Figure 7

G-CSF level in serum after subcutaneous (S.C.) administration of G-CSF and 3DHSA-G-CSF. The concentration-time curve of 3DHSA-G-CSF is superior than that of G-CSF. Solid black circle means G-CSF. Black hollow circle means 3DHSA-G-CSF. Each point represents the mean value, and error bars represent SD of the mean (n = 3). Data are representative of three independent experiments with similar result.