title: Strategy for linker selection to enhance refolding and bioactivity of VAS-TRAIL fusion protein based on inclusion body conformation and activity.
abstract: A bifunctional fusion protein, VAS-TRAIL, was designed for superior therapeutic efficacy by combining anti-angiogenesis activity with tumor-selective apoptosis activity. The protein was expressed as inclusion body (IB) in Escherichia coli. To enhance refolding yield and bioactivity, four fusions were constructed with different linkers (no linker, flexible linker, rigid linker, and helix-forming linker). A novel linker selection strategy based on IB conformational quality and activity was applied to predict the suitable linker. The conformational quality and activity of VAS-TRAIL IBs were analyzed by ATR-FTIR and cytotoxicity assay, respectively. Results demonstrated that aggregated VRT (fusion with rigid linker) contained the highest native-like β structure content and retained part of the expected activity, namely, cytotoxicity activity on tumor cells. This finding suggested that the rigid linker was the most suitable candidate. Further results of in vitro refolding and subsequent circular dichroism and activity assay of four refolded fusions were significantly correlated with the predictions. Refolding of VRT yielded more soluble proteins containing the expected secondary structure and the highest bioactivity compared with that of other fusions. Our research may offer an efficient method for the high-throughput design of aggregated-prone therapeutic fusion protein.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jiying
authors.initials: J
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Liying
authors.initials: L
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Sun
authors.affiliation: Shanghai Gebaide Biotechnical Co., Ltd., Shanghai 201403, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Qiu
authors.affiliation: Shanghai Gebaide Biotechnical Co., Ltd., Shanghai 201403, China.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai 200237, China. Electronic address: ylshen@ecust.edu.cn.
authors.forename: Yaling
authors.initials: Y
authors.lastname: Shen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D037282:Calreticulin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D037282:Calreticulin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C121986:vasostatin
keywords: Inclusion body quality; Linker engineering; Protein refolding; VAS-TRAIL fusion protein