Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate cell-matrix composition and are also involved in processing various bioactive molecules such as cell-surface receptors, chemokines, and cytokines. Our group recently reported that MMP-3, -8, and -9 are upregulated during microglial activation and play a role as proinflammatory mediators (Lee et al., 2010, 2014). In particular, we demonstrated that MMP-8 has tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α and that inhibition of MMP-8 inhibits TACE activity. The present study was undertaken to compare the effect of MMP-8 inhibitor (M8I) with those of inhibitors of other MMPs, such as MMP-3 (NNGH) or MMP-9 (M9I), in their regulation of TNF-α activity. We found that the MMP inhibitors suppressed TNF-α secretion from lipopolysaccharide (LPS)-stimulated BV2 microglial cells in an order of efficacy: M8I>NNGH>M9I. In addition, MMP inhibitors suppressed the activity of recombinant TACE protein in the same efficacy order as that of TNF-α inhibition (M8I>NNGH>M9I), proving a direct correlation between TACE activity and TNF-α secretion. A subsequent pro-TNF-α cleavage assay revealed that both MMP-3 and MMP-9 cleave a prodomain of TNF-α, suggesting that MMP-3 and MMP-9 also have TACE activity. However, the number and position of cleavage sites varied between MMP-3, -8, and -9. Collectively, the concurrent inhibition of MMP and TACE by NNGH, M8I, or M9I may contribute to their strong anti-inflammatory and neuroprotective effects.

Frontiers in Bioengineering and Biotechnology3区 · 工程技术

Process Intensification for an Insect Antimicrobial Peptide Elastin-Like Polypeptide Fusion Produced in Redox-Engineered Escherichia coli

3区 · 工程技术

ArticleOA

作者: Maguire, Nicolas ; Gerlach, Doreen ; Schäfer, Johannes ; Joachim, Mathias ; Czermak, Peter

Peptides and proteins containing disulfide bonds can be produced in Escherichia coli by targeting the oxidizing periplasm, co-expressing isomerases or chaperons, refolding from inclusion bodies, or by using redox-engineered E. coli strains. Thus far, protein expression in glutathione reductase and thioredoxin reductase deficient (Δgor ΔtrxB) E. coli strains has required a complex medium. However, a chemically defined medium suitable for large-scale production would be preferable for industrial applications. Recently, we developed a minimal medium supplemented with iron (M9i) for high-density cultivation using E. coli Rosetta gami B(DE3)pLysS cells. Here we show that M9i is suitable for the production of insect metalloproteinase inhibitor (IMPI), which contains five disulfide bonds, in the same E. coli strain. We demonstrated the scalability of the new fed-batch process by combining the scale-up criteria of constant dissolved oxygen (DO) and matching volumetric power inputs (P/V) at the borders of the stirrer cascade. Process intensification was achieved by investigating production feed rates and different induction times. We improved product titers by ~200-fold compared to the standard process in complex medium while maintaining the activity of the IMPI protein. Our results show for the first time that it is possible to produce active proteins containing multiple disulfide bonds in a Δgor ΔtrxB E. coli strain using M9i medium. The success of scale-up and process intensification shows that the industrial production of complex recombinant proteins in such strains using chemically defined M9i minimal medium is feasible.

100 项与 M9i 相关的药物交易

登录后查看更多信息