Abstract
BackgroundIdentification 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).ResultsDicistronic-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.ConclusionThe 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.
Highlights
Escherichia coli is a suitable host for the initial screening of recombinant protein expression owing to easy genetic manipulation, inexpensive culture, and rapid growth [1, 2]
We developed a simple screening system at a micro-scale that can be used for rapid screening of high-expressing colonies in E. coli
The inoculation was examined at different volumes of the newly transformed suspension on Luria Bertani (LB) agar plates containing both selective antibiotics to obtain a single clone in each well of 96-microplate
Summary
Escherichia coli is a suitable host for the initial screening of recombinant protein expression owing to easy genetic manipulation, inexpensive culture, and rapid growth [1, 2]. The screening with the SDS-PAGE technique is laborintensive, time-consuming, and usually restricted to a limited number of colons that may lead to missing high expressing colons The emerging techniques, such as fluorescence-activated cell sorting (FACS) and robotic platforms, facilitate the colony selection program. Expensive equipment and special expertise might not be available in every research laboratory In these platforms, even with automation, the protein production pipeline often consists of several steps that have to be linked together and require manual interventions. These platforms require high-resolution imaging systems to monitor the colonies growing on the agar surface and discriminate them based on user-selectable criteria, such as diameter and roundness. 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)
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