We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides.

先前我们已经表明，小的金属结合蛋白CusF3H +和SmbP可以用作融合蛋白，用于在大肠杆菌中表达和纯化重组蛋白。由于它们的大小都很小（均为10 kDa左右），因此它们适合于肽的生产，以避免在去除标签的最后纯化步骤后产量不高。Bin1b是在大鼠的附睾中发现的β-防御素，已显示具有抗菌活性。用于在大肠杆菌中表达这种抗菌肽的先前方法涉及将肽表达为包涵体，然后通过第二质粒进行体外重折叠或补充使肽在细胞质中正确折叠所必需的蛋白质。在这里，我们开发了一种放弃这些方法的方法，而是使用融合蛋白CusF3H +或SmbP和大肠杆菌菌株SHuffle获得了含有成熟Bin1b肽的可溶性重组蛋白。使用IMAC色谱法纯化重组蛋白，然后用肠激酶裂解，从Bin1b中分离融合蛋白。如前所述，纯化的肽显示出对大肠杆菌的抗菌活性。此外，我们还测试了其对革兰氏阳性细菌的抗菌活性金黄色葡萄球菌，并发现Bin1b也能够抑制这种细菌的生长。总之，我们开发了一种实用的方法，用于使用融合蛋白CusF3H +和SmbP在大肠杆菌中表达和纯化生物活性Bin1b肽。该方法可以进一步用于产生更具生物活性的肽。