Centre for Innovation in Infectious Disease Research, Education and Training (CIIDRET), University of Delhi South Campus, New Delhi, India.
Department of Biochemistry, University of Delhi South Campus, New Delhi, India.
PLoS One. 2020 Jul 23;15(7):e0235853. doi: 10.1371/journal.pone.0235853. eCollection 2020.
PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.
基于 PCR 的已注释基因扩增允许使用经典和基于重组的克隆技术在全基因组范围内构建表达克隆。然而，用于下游应用的蛋白质的全基因组规模表达和纯化通常受到以下挑战的限制：表达不佳、溶解度低、多结构域蛋白质的尺寸大等。或者，表达载体中的 DNA 片段文库可以作为蛋白质片段的来源，每个片段都包含其全长蛋白质对应物的一个功能。然而，随机的 DNA 片段化和克隆导致只有 18 个克隆中的 1 个处于正确的开放阅读框（ORF），从而降低了整个系统的效率。这就需要在表达蛋白质片段之前选择正确的 ORF。本文描述了一种用于 DNA 片段文库的高效 ORF 选择系统，该系统基于分裂β-内酰胺酶蛋白片段互补。该系统的设计允许使用无限制酶克隆策略将选定的 DNA 片段文库无缝转移到任何下游载体系统中。该策略已应用于使用模型构建体选择 ORF，以显示近 100%选择编码正确 ORF 的克隆。该系统进一步通过构建结核分枝杆菌 30 个基因的 ORF 选择 DNA 片段文库得到验证。此外，我们已经成功地在大肠杆菌中证明了 ORF 选择的蛋白质片段的胞质表达。
