Department of Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
Microb Cell Fact. 2021 Dec 11;20(1):223. doi: 10.1186/s12934-021-01711-2.
Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27).
Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze-thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking.
The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.
在重组蛋白开发的上游过程中，鉴定高表达集落是主要关注点之一。筛选高产集落的常用方法是 SDS-PAGE，这是一个费力且耗时的过程，基于随机和定性的方法。本研究描述了一种快速筛选系统的设计和开发，该系统由包含报告基因（增强型绿色荧光蛋白，eGFP）、蛋白模型（葡萄球菌激酶，SAK）的双顺反子表达系统和包含热休克蛋白 27（Hsp27）的自诱导系统组成。
双顺反子-自诱导系统在 5ml 和 1L 培养体积中成功表达了 eGFP 和 SAK。在 6 小时内通过荧光信号鉴定出高表达集落。此外，通过放射状酪蛋白溶解和显色法分别半定量和定量确认了蛋白模型的生物活性。eGFP 荧光强度与 SAK 活性之间存在直接相关性。通过 Pearson 相关性和线性回归分别确认了两个基因之间的表达相关性和线性。此外，还确定了精密度、检测限（LOD）和定量限（LOQ）。eGFP 和 SAK 的表达在四次冻融循环中保持稳定。此外，所开发的方案表明，转化体可以直接接种到培养物中，节省时间并减少集落挑选的易错步骤。
该开发的系统适用于大多数研究实验室中快速筛选高表达集落。该系统可用于研究在大肠杆菌中表达的其他重组蛋白，具有自动化和在更大规模使用的潜力。
