Background: The engineered chimeric peptides including functional multi-epitope structures fused by various peptide linkers are widely applied in biotechnological research to improve the expression level and biological activity of chimera.

Objective: The aim of our study was to evaluate the effect of helical and flexible linkers on solubility, expression level and folding of multi-epitope chimera containing four epitopes of Human T Lymphotropic Virus Type 1 (HTLV-1).

Methods: For this purpose, the chimera sequences connected by the helical or flexible linker were inserted into different plasmid vectors and expressed in E. coli strains. The expressed products were analyzed using SDS-PAGE and Western blot techniques. Additionally, the molecular modeling study of the chimera with helical or flexible linker was performed using iterative threading assembly refinement (I-TASSER) to attain their three-dimensional structures.

Results: Comparison of the chimera expression indicated that the insertion of a flexible (GGGGS)3 linker among chimera epitopes could significantly enhance the level of expression, whereas, the low-level of chimera expression was observed for chimera containing the contiguous helical (EAAAK)5 linker. According to the results of sequence alignment and plasmid stability test, the structure and function of a consecutive helical linker among chimera epitopes were similar to porins as the outer-membrane pore-forming proteins. The molecular modeling results confirmed our experimental study.

Conclusion: This investigation illustrated the key role of linker design in determining the expression level of multi-epitope chimera and conformational folding.

背景：经过工程改造的嵌合肽，包括通过各种肽接头融合的功能性多表位结构，被广泛应用于生物技术研究中，以提高嵌合体的表达水平和生物学活性。

目的：我们的研究目的是评估螺旋和柔性接头对含有1型人T淋巴病毒（HTLV-1）四个表位的多表位嵌合体的溶解度，表达水平和折叠的影响。

方法：为此，将通过螺旋或柔性接头连接的嵌合序列插入不同的质粒载体中，并在大肠杆菌菌株中表达。使用SDS-PAGE和蛋白质印迹技术分析表达的产物。此外，使用迭代螺纹装配细化（I-TASSER）进行具有螺旋或柔性接头的嵌合体的分子建模研究，以获得其三维结构。

结果：嵌合体表达的比较表明，在嵌合体表位之间插入柔性（GGGGS）3接头可以显着提高表达水平，而对于包含连续螺旋（EAAAK）的嵌合体，则观察到了较低的嵌合体表达水平。 5个链接器。根据序列比对和质粒稳定性测试的结果，嵌合表位之间连续螺旋接头的结构和功能与作为外膜成孔蛋白的孔蛋白相似。分子建模结果证实了我们的实验研究。

结论：这项研究说明了接头设计在确定多表位嵌合体的表达水平和构象折叠中的关键作用。