Buforin I is a cationic antimicrobial peptide derived from histone proteins. It is a 39-amino acid peptide with a high proportion of basic residues, such as arginine and lysine, contributing to its strong positive charge. The peptide exhibits a broad spectrum of activity against various Gram-positive and Gram-negative bacteria and fungal strains. Structural analyses, including FTIR and modeling studies, indicate that Buforin I possesses an α-helical conformation, which is stabilized by the presence of a proline residue that introduces a hinge-like structure. This conformation is considered important for its functional interaction with microbial membranes.

Recombinant expression of Buforin I has been achieved in the periplasmic space of Escherichia coli using a pET-based system. The addition of a poly-histidine tag to the peptide's N-terminus facilitates purification via nickel affinity chromatography. Comparative studies between the native and His-tagged recombinant Buforin I reveal minimal differences in key physicochemical properties, including isoelectric point, net charge, and in silico stability parameters such as root mean square fluctuation (RMSF) and radius of gyration (Rg). The structural integrity and secondary α-helical conformation of the peptide are preserved following the His-tag fusion, as confirmed by spectroscopic methods.

• Roshanak, S., Yarabbi, H., Shahidi, F. et al. Effects of adding poly-histidine tag on stability, antimicrobial activity and safety of recombinant buforin I expressed in periplasmic space of Escherichia coli. Sci Rep 13, 5508 (2023). https://doi.org/10.1038/s41598-023-32782-3

三氟乙酸（Trifluoroacetic acid，简称 TFA）是高效液相色谱（简称 HPLC）纯化过程中常用的反离子。TFA 的存在可能会对肽的净重量、外观及溶解度产生影响，具体如下：

对净重量的影响：TFA 盐会增加产物的总质量。在大多数情况下，肽的含量占总重量的 80% 以上，剩余部分则为 TFA。

对溶解度的影响：TFA 盐通常能提高肽在水溶液中的溶解度。

在生物检测中的影响：对于大多数标准的体外检测，残留的 TFA 水平一般不会造成干扰。但需注意，在高灵敏度的细胞研究或生化研究中，需关注其存在可能带来的影响。

干品多肽的重量中不仅仅包含多肽，还包含有一些非肽的组份，如水、被吸收的溶剂、配位离子和盐等。肽的净含量是指肽在其中的重量百分比，这个百分比的数值范围很大，可能从50%到90%，取决于纯度、序列以及合成和纯化的方法，不要将肽的净含量和肽的纯度混为一谈，他们是两个完全不同的概念。纯度通常是通过HPLC测定的。纯度定义的是多肽样品中含正确序列的组分的百分比，而肽的净含量是指样品中肽类物质相对于总物质所占的百分比，肽的净含量通常是氨基酸组分分析或紫外分光法测定的，这个信息主要是在一些对肽的浓度很敏感的实验中，对计算肽的浓度是很重要的。