Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System
AbstractHuman macrophage inflammatory protein 3α (MIP-3α), also known as CCL20, is a 70 amino acid chemokine that selectively binds and activates chemokine receptor 6 (CCR6). This chemokine is responsible for inducing the migration of immature dendritic cells, effector, or memory T-cells, and B-cells. Moreover, the MIP-3α protein has been shown to display direct antimicrobial, antiviral and antiprotozoal activities. Because of the potential therapeutic uses of this protein, the efficient production of MIP-3α is of great interest. However, bacterial recombinant production of the MIP-3α protein has been limited by the toxicity of this extremely basic protein (pI 9.7) toward prokaryotic cells, and by solubility problems during expression and purification. In an attempt to overcome these issues, we have investigated the bacterial recombinant expression of MIP-3α by using several common expression and fusion tags, including 6× histidine (His), small ubiquitin modifier protein (SUMO), thioredoxin (TRX), ketosteroid isomerase (KSI), and maltose binding protein (MBP). We have also evaluated a recently introduced calmodulin (CaM)-tag that has been used for the effective expression of many basic antimicrobial peptides (AMPs). Here, we show that the CaM fusion tag system effectively expressed soluble MIP-3α in the cytoplasm of Escherichia coli with good yields. Rapid purification was facilitated by the His-tag that was integrated in the CaM-fusion protein system. Multidimensional nuclear magnetic resonance (NMR) studies demonstrated that the recombinant protein was properly folded, with the correct formation of disulfide bonds. In addition, the recombinant MIP-3α had antibacterial activity, and was shown to inhibit the formation of Pseudomonas aeruginosa biofilms. View Full-Text
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Ramamourthy, G.; Arias, M.; Nguyen, L.T.; Ishida, H.; Vogel, H.J. Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System. Microorganisms 2019, 7, 8.
Ramamourthy G, Arias M, Nguyen LT, Ishida H, Vogel HJ. Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System. Microorganisms. 2019; 7(1):8.Chicago/Turabian Style
Ramamourthy, Gopal; Arias, Mauricio; Nguyen, Leonard T.; Ishida, Hiroaki; Vogel, Hans J. 2019. "Expression and Purification of Chemokine MIP-3α (CCL20) through a Calmodulin-Fusion Protein System." Microorganisms 7, no. 1: 8.
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