Protein expression and purification · 2026
Engineering of dual-responsive elastin-like polypeptide (ELP) systems for precision delivery of calcitonin gene-related peptide (CGRP): Impact of chain length on performanc.
СтатьяНазвание и аннотация: Перевод пока не подготовлен
Обработка статьи · этапы, ошибки и управление
Загрузка состояния…
Ошибки и ограничения
История · было → стало
Состояние обновляется каждые 5 секунд, пока блок открыт. Процент показывается только там, где известен объём работы. Пауза останавливает продолжение этапа; текущий запрос к источнику может завершиться.
Zhao Qi, Yang Lei, Pang Bowen, Wei Pengfei, Fang Guanqi, Chen Shuo, Chen Ting
Аннотация
BACKGROUND: Calcitonin gene-related peptide (CGRP) holds significant potential for tissue regeneration and bone repair; however, its clinical application is limited by a rapid systemic half-life and the difficulty of maintaining localized therapeutic concentrations. This study aimed to design and characterize a dual-responsive fusion protein system, utilizing Elastin-Like Polypeptides (ELPs) as a smart delivery vehicle to achieve spatiotemporal control of CGRP release.
METHODS: We engineered four fusion protein variants ELPn-CGRP, where n = 20, 40, 60, 80) by modulating the ELP chain length to tune the phase transition temperature (Tt). A Matrix Metalloproteinase-9 (MMP-9) specific cleavage site was integrated between the ELP carrier and the CGRP payload to enable microenvironment-responsive release. The recombinant proteins were expressed in E. coli, and a synergistic purification strategy-combining temperature-induced Inverse Transition Cycling (ITC) with nickel-affinity chromatography-was developed and optimized.
RESULTS: Characterization of the biophysical properties revealed that the Tt of the ELP60-CGRP variant aligns closely with human physiological temperature, ensuring stable localized coacervation upon administration. The optimized dual-purification protocol significantly enhanced protein purity and reduced host-cell protein contamination compared to conventional methods. Furthermore, in vitro assays confirmed that the fusion proteins remain sensitive to MMP-9, effectively releasing functional CGRP in response to enzymatic triggers.
CONCLUSIONS: This work demonstrates a robust bio-engineering approach for the production and delivery of neuropeptides. The ELPn-CGRP platform leverages a dual-responsive "catch-and-release" mechanism-achieving localized sequestration via thermal phase transition and site-specific release through enzymatic cleavage. This system presents a robust strategy for high-concentration localized administration in regenerative medicine.
METHODS: We engineered four fusion protein variants ELPn-CGRP, where n = 20, 40, 60, 80) by modulating the ELP chain length to tune the phase transition temperature (Tt). A Matrix Metalloproteinase-9 (MMP-9) specific cleavage site was integrated between the ELP carrier and the CGRP payload to enable microenvironment-responsive release. The recombinant proteins were expressed in E. coli, and a synergistic purification strategy-combining temperature-induced Inverse Transition Cycling (ITC) with nickel-affinity chromatography-was developed and optimized.
RESULTS: Characterization of the biophysical properties revealed that the Tt of the ELP60-CGRP variant aligns closely with human physiological temperature, ensuring stable localized coacervation upon administration. The optimized dual-purification protocol significantly enhanced protein purity and reduced host-cell protein contamination compared to conventional methods. Furthermore, in vitro assays confirmed that the fusion proteins remain sensitive to MMP-9, effectively releasing functional CGRP in response to enzymatic triggers.
CONCLUSIONS: This work demonstrates a robust bio-engineering approach for the production and delivery of neuropeptides. The ELPn-CGRP platform leverages a dual-responsive "catch-and-release" mechanism-achieving localized sequestration via thermal phase transition and site-specific release through enzymatic cleavage. This system presents a robust strategy for high-concentration localized administration in regenerative medicine.