Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

Shuqiang Zhao,Hong Tian,Wenbing Yao,Di Cai,Xiaofei Chen,Xiangdong Gao,Yu Zhang

doi:10.1155/2013/107238

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 pPICZαA 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.

Highlights

Human Granulocyte colony stimulating factor (G-CSF) gene was first reported in 1986, and G-CSF is a 174 amino acid glycoprotein belonging to the family of hematopoietic cytokine [1]
Six mice of per group were subjected with saline, G-CSF, and 3DHSA-G-CSF, and twenty-four hours after administration, white blood cell (WBC) counts were determined with Sysmex NE-8000
The full length 3DHSA-G-CSF gene of 1164 bp in size was obtained through overlap PCR, and the purified fusion protein gene was inserted into the expression vector pPICZαA to generate a recombinant plasmid pPIC-3DHSAG-CSF

Summary

Introduction

Human Granulocyte colony stimulating factor (G-CSF) gene was first reported in 1986, and G-CSF is a 174 amino acid glycoprotein belonging to the family of hematopoietic cytokine [1]. In order to prolong the half-life of G-CSF, we genetically fused it with 3DHSA and successfully expressed the 3DHSA-G-CSF fusion protein in P. pastoris GS115. To our knowledge, this is the first report of 3DHSAG-CSF expression in any expression system. The biological activity and pharmacokinetic property of the purified fusion protein were studied utilizing neutropenia model mice and normal mice, and the results indicated that 3DHSA-GCSF may be considered as a drug candidate of neutropenia for further research and development

Materials and Methods

Results

Discussions