Prokaryotic Soluble Expression, Purification, Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11

doi:10.13523/j.cb.2212024

China Biotechnology

2023, Vol. 43

Issue (7): 44-52 DOI: 10.13523/j.cb.2212024

Prokaryotic Soluble Expression, Purification, Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11

Huan MENG,Yi-fan SU,Sa WANG,Shu-wen ZHANG,Yong-xiang ZHENG,Rong YU,Chun ZHANG^**(

)

Key Laboratory of Drug Targeting and Drug Delivery System (Ministry of Education), West China School of Pharmacy, Sichuan University, Chengdu 610041, China

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Abstract

Objective: To improve the soluble expression level of human interleukin-11 in prokaryotic expression system and enhance its circulating half-life in vivo. Methods: The recombinant protein expression vector (PBV220-ABD-rhIL11) was constructed, and it was successfully expressed in prokaryotic expression system in soluble form. Based on hydrophobic chromatography, ion exchange chromatography and gel chromatography, a method for purification of ABD-rhIL-11 from the supernatant of bacteria was successfully established. The circular dichroism spectrum and fluorescence emission spectrum analysis confirmed that the spatial structure of the purified ABD-rhIL-11 was folded correctly. In vivo and in vitro activity and pharmacodynamic evaluation was carried out. Results: The purity of recombinant fusion protein was higher than 95%. The results of in vitro binding experiments showed that ABD-rhIL-11 could rapidly bind to human serum albumin. The results of cell proliferation experiment showed that the activity of ABD-rhIL-11 was equivalent to that of the control group. Pharmacokinetic results showed that the fusion protein could significantly prolong the circulating half-life of interleukin-11 in rats. The results of platelet count test showed that ABD-rhIL-11 could effectively stimulate platelet proliferation in rats. Conclusion: The results of this study would provide a new idea for the long-term design of interleukin-11 drugs based on albumin binding peptide fusion strategy.

Key words： Human interleukin-11 Albumin binding peptide Soluble expression Platelet proliferation

Received: 18 December 2022 Published: 03 August 2023

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	Huan MENG
	Yi-fan SU
	Sa WANG
	Shu-wen ZHANG
	Yong-xiang ZHENG
	Rong YU
	Chun ZHANG

Cite this article:

Huan MENG, Yi-fan SU, Sa WANG, Shu-wen ZHANG, Yong-xiang ZHENG, Rong YU, Chun ZHANG. Prokaryotic Soluble Expression, Purification, Characterization and Preliminary Activity Evaluation of Recombinant Albumin Binding Peptide Fused with Human Interleukin-11. China Biotechnology, 2023, 43(7): 44-52.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2212024 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I7/44

Fig.1 Ide.pngication of the expression level and expression form in Escherichia coli using SDS-PAGE

Fig.2 Chromatograms of the purification of the ABD-IL-11 and SDS-PAGE analysis of eluted peaks (a) Chromatograms of the purification of the ABD-IL-11 through capto butyl (b) Chromatograms of the purification of the ABD-IL-11 through capto S (c) Chromatograms of the purification of the ABD-IL-11 through superdex 75 (d) 12% SDS-PAGE analysis eluted peaks of capto butyl (e) 12% SDS-PAGE analysis eluted peaks of capto S (f) 12% SDS-PAGE analysis eluted peaks of superdex 75

Fig.3 Binding of the ABD-IL-11 to human serum albumin in vitro

Fig.4 Characterization of ABD-IL-11 (a) RP-HPLC (b) CD (c) SEC-HPLC (d) DLS

Fig.5 Relative antibody binding affinity and cell proliferation of the ABD-IL-11 and IL-11 (a) Antibody relative binding capacity (b) Cell viability

Fig.6 Pharmacokinetics of the conjugate in SD male rats

Fig.7 Rat blood samples (50-fold dilution) microscopic 100 μm field of view (a) PBS control group (b) IL-11 reference group (c) ABD-IL-11 sample set

Fig.8 Platelet count result of rat blood


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