Preparation, characterization and in vitro bioactivity of N-terminally PEGylated staphylokinase dimers |
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Authors: | Ruyan Liu Dongxia LiJun Wang Rui Qiu Qixun LinGuifeng Zhang Guanghui MaZhiguo Su Tao Hu |
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Institution: | a National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 Bei-Er-Tiao Street, Haidian District, Beijing 100190, China b Graduate School, Chinese Academy of Sciences, Beijing 100190, China c College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China |
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Abstract: | PEGylation can improve the therapeutic efficacy of proteins by increasing serum half-life of proteins and reducing immunogenicity and antigenicity. However, PEGylation results in a substantial loss of the bioactivity of proteins due to the steric hindrance of polyethylene glycol (PEG). Dimerization of the proteins is an efficient approach to increase the bioactivity of the PEG-protein conjugates. Here, staphylokinase (SAK) was used due to its therapeutic potential for coronary thrombolysis. SAK dimers (dSAK) were prepared by engineering cysteine residue at the C-terminus of SAK and dimerization of the cysteine residue with 1,4-bismaleimidobutane. PEG aldehyde was used for site-specific PEGylation of dSAK at one of its two N-termini. Structural analysis indicated that dimerization of SAK can decrease the steric hindrance of PEG and increase the binding affinity of PEG-SAK to plasminogen. Dimerization of SAK increased the relative bioactivity of PEG-SAK from 39.0% to 62.0%. Therefore, site-specifically PEGylated dSAK at one of its two N-termini has higher bioactivity than the N-terminal PEGylated SAK. |
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Keywords: | PEG polyethylene glycol SAK staphylokinase dSAK SAK dimers PEG-SAK the monoPEGylated SAK PEG-dSAK the monoPEGylated dSAK BMB 1 4-bismaleimidobutane SEC size exclusion chromatography CD circular dichroism ANS 1-anilino-8-naphthalene sulfonic acid SPR surface plasmon resonance |
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