類澱粉蛋白纖維(amyloid fibrils)在醫學上為導致的阿茲海默和帕金森氏症的主要因素之一，然而類澱粉蛋白的獨特的自組裝特性可用於應用，功能性類澱粉蛋白亦存在於自然界生物細胞中，如由桿菌產生的類澱粉蛋白纖維，稱為捲曲纖維(curli fiber)。類澱粉蛋白傾向自組裝為具有奈米孔洞之高表面積類澱粉纖維網絡，其性質非常穩定，可以抵抗蛋白水解酶的水解和其他惡劣條件。此外，其高生物相容性使其廣泛用於藥物輸送，生物傳感器和組織工程的奈米纖維材料等各個領域。
大腸桿菌或沙門氏菌的胞外纖維，稱為Csg系統。CsgA是捲曲纖維的主要亞基蛋白，它們在細胞質合成，然後以未折疊蛋白之形式運輸到細胞表面，在細胞表面自組裝後成為細胞外類澱粉蛋白纖維。已有許多研究將功能性胜肽或酵素蛋白與CsgA蛋白融合，經細胞表現生產後，此融合蛋白自組裝形成類澱粉蛋白纖維，而可成為酵素或功能性蛋白之固定化平台。
本研究使用基因工程來生產CsgA融合蛋白，CsgA-Ag4和CBD-CsgA-Ag4，藉由大腸桿菌表達，並利用CsgA本身的自組裝能力和其與各種親疏水性材料表面之作用力將功能性胜肽固定化於表面，再藉由與之融合的銀離子結合胜肽(silver binding peptide)還原銀離子形成奈米銀使材料表面達到抗菌的效果，自組裝功能性奈米纖維具有一定的機械強度與穩定性以抵抗外在環境的變化。研究結果顯示CsgA-Ag4與CBD-CsgA-Ag4皆能在大腸桿菌系統中表達，且維持其功能進行自組裝並還原銀離子。CsgA-Ag4和CBD-CsgA-Ag4銀還原量差別不大，ICP結果顯示CBD-CsgA-Ag4銀還原量只高於CsgA-Ag4 0.81%，而每1 mg的CsgA融合蛋白能還原約17.7 ppm的銀離子。兩者抗菌效果相對於基材本身皆展示出99%以上的抗菌效果，而CsgA-Ag4相較於CBD-CsgA-Ag4有較好的抗菌表現。

Amyloid fibrils formation in the tissue is one of the major factors which caused Alzheimer's disease and Parkinson's disease in human beings. However, the unique self-assemble property of amyloid can have some bio-applications. This type of proteins produced by Bacillus and are called Curli fiber. Amyloid are easy to self-aggregate to a fiber network which has a high surface area with nanopores. The network is very stable and can resist the hydrolysis of proteolytic enzymes and other harsh conditions. In addition, the high biocompatibility property make it widely used in various fields such as drug delivery, biosensors and tissue engineering’s nano-fiber material.
The extracellular fiber of Escherichia coli or Salmonella, called Csg system are some parts of Curli fiber. CsgA are the main subunit proteins of curli fiber, which are synthesized in the cytoplasm in the form of unfolded proteins and then transported to the cell surface. Then they self-assemble on the cell surface and become extracellular amyloid fibrils. There are many previous reports about CsgA protein fused with some functional peptides or enzymes. After protein expression in cells, the fusion protein self-assembles and become amyloid fibrils, which can be used as immobilized platform for enzymes or functional proteins.
In this work, we used genetic engineering to produce recombinant CsgA and its fusion with some functional peptides such as CsgA-Ag4 and CBD-CsgA-Ag4. All proteins have been expressed using E. coli as the host. CsgA and its fusions can self-assemble on both hydrophilic and hydrophobic surfaces due to CsgA’s characteristic of self adhesion. The fusion proteins CsgA-Ag4 and CBD-CsgA-Ag4 also had ability to bind with silver ions and reduced into silver nanoparticles via silver binding peptide (Ag4) and making the substrates surface antibacterial.
The self-assembled functional nanofibers had a good mechanical strength and stability to resist the changes of the external environment. The results demonstrate that both CsgA-Ag4 and CBD-CsgA-Ag4 could be cloned and expressed in the E. coli system. The proteins could properly self-assembled on the substrates such as glass, silicone, and PC (polycarbonate) and reduced the binding silver ions. After compared both of fusion proteins, only small amount difference in silver reduction effect. ICP result showed the reduction amount of silver ions in the CBD-CsgA-Ag4 was only 0.81% higher than the CsgA-Ag4. The CsgA fusion proteins showed 17.7 ppm reduced amount of silver ion per mg protein. Both CsgA fusion proteins showed approximately 99% antibacterial effect compared to the bare substrate. In conclusion, CsgA-Ag4 had better antibacterial performance compared to CBD-CsgA-Ag4.

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