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Published on: July 21, 2021
Homologous chaperone-mediated soluble production of Mfp-3 and its application in atopic dermatitis therapy
Kundong He1, Yu Wang1, Weizhi Tian1
1College of Food Science and Light Industry, Nanjing Tech University, Nanjing, 211816, China; State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, 211816, China.
Abstract:
Mussel foot proteins (Mfps) are a class of specialized adhesive proteins synthesized by marine mussels. Among these, type 3 mussel foot protein (Mfp-3) has tremendous application potential as a cosmetic and medical raw material, owing to its excellent antioxidant and anti-inflammatory properties. However, the low efficiency of the natural extraction method (only 1 mg of protein can be obtained from 10,000 mussels) has hindered the industrial application. Heterologous expression via genetic recombination offers a reliable alternative. However, a major challenge is that recombinant expression frequently results in misfolded, biologically inactive protein aggregates. In this study, we identified endogenous molecular chaperones in Mytilus galloprovincialis and successfully constructed a strain that co-expresses Mfp-3 and these chaperone proteins, thereby achieving the soluble expression of Mfp-3. Furthermore, through optimization of fermentation conditions, the soluble yield of Mfp-3 in high-density fermentation using a 5-L fermenter reached 713 mg/L. Meanwhile, following in vitro tyrosinase modification, the L-3,4-dihydroxyphenylalanine (DOPA) content of Mfp-3 reached 4.45%, corresponding to a modification rate of 22.26%. On this basis, this study also demonstrated that soluble Mfp-3 possesses excellent cell migration-promoting ability. Moreover, it exhibited superior biological activity in the treatment of atopic dermatitis by reducing levels of pro-inflammatory cytokines and immunoglobulins. This research provides a new strategy for the production of soluble Mfp-3 in Escherichia coli and offers a reference for the large-scale production of soluble Mfps.
Insights
Researchers developed a method for soluble expression of mussel foot protein-3 (Mfp-3) using co-expression with molecular chaperones in E. coli. This strategy significantly improves Mfp-3 yield and biological activity for cosmetic and medical applications.
Area of Science:
- Biomaterials Science
- Protein Engineering
- Marine Biotechnology
Background:
- Mussel foot proteins (Mfps), particularly Mfp-3, show promise for cosmetic and medical uses due to antioxidant and anti-inflammatory properties.
- Natural extraction of Mfp-3 is inefficient, yielding only 1 mg per 10,000 mussels, hindering industrial application.
- Recombinant expression of Mfp-3 often leads to misfolded, inactive protein aggregates, posing a significant challenge.
Purpose of the Study:
- To develop a method for the soluble and biologically active production of Mfp-3.
- To overcome challenges associated with recombinant protein misfolding and aggregation.
- To establish a scalable and efficient production strategy for Mfp-3.
Main Methods:
- Identification of endogenous molecular chaperones in Mytilus galloprovincialis.
- Construction of a genetically engineered E. coli strain for co-expression of Mfp-3 and identified chaperones.
- Optimization of high-density fermentation conditions in a 5-L fermenter.
- In vitro tyrosinase modification to increase L-3,4-dihydroxyphenylalanine (DOPA) content.
Main Results:
- Achieved soluble expression of Mfp-3 by co-expressing with molecular chaperones.
- Obtained a soluble Mfp-3 yield of 713 mg/L through optimized high-density fermentation.
- Increased Mfp-3's L-3,4-dihydroxyphenylalanine (DOPA) content to 4.45% (22.26% modification rate) via tyrosinase treatment.
- Demonstrated soluble Mfp-3's ability to promote cell migration and its efficacy in reducing inflammatory markers in atopic dermatitis models.
Conclusions:
- Co-expression with molecular chaperones is an effective strategy for achieving soluble Mfp-3 production in E. coli.
- Optimized fermentation and post-expression modification enhance Mfp-3 yield and biological functionality.
- This research provides a viable pathway for the large-scale industrial production of Mfp-3 and other Mfps.
