Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha2b fusion protein by linker engineering.
Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha2b (IFN-alpha2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha2b was ascribed to the structural disturbance between HSA and IFN-alpha2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.
Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China.
Hong Liang
HL
Zhao
Xue Qin
XQ
Yao
Chong
C
Xue
Yang
Y
Wang
Xiang Hua
XH
Xiong
Zhi Min
ZM
Liu
D000595:Amino Acid Sequence; D000998:Antiviral Agents; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics*
D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin