High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Kim, Young Min; Ganesan, Prabhakar; Ihee, Hyot Cherl

doi:10.1002/pro.2286

Cited by 14 publications

(8 citation statements)

References 66 publications

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“…In addition to its biological interest, PYP is a popular model signaling protein due to its small size. As such, PYP has found use as a prototype biosensor [3] , and in procedures for quantifying protein expression [4] . In addition PYP and its circularly permuted variants have been used as a photo switch [5] – [7] .…”

Section: Introductionmentioning

confidence: 99%

Multiscale Approach to the Determination of the Photoactive Yellow Protein Signaling State Ensemble

et al. 2014

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The nature of the optical cycle of photoactive yellow protein (PYP) makes its elucidation challenging for both experiment and theory. The long transition times render conventional simulation methods ineffective, and yet the short signaling-state lifetime makes experimental data difficult to obtain and interpret. Here, through an innovative combination of computational methods, a prediction and analysis of the biological signaling state of PYP is presented. Coarse-grained modeling and locally scaled diffusion map are first used to obtain a rough bird's-eye view of the free energy landscape of photo-activated PYP. Then all-atom reconstruction, followed by an enhanced sampling scheme; diffusion map-directed-molecular dynamics are used to focus in on the signaling-state region of configuration space and obtain an ensemble of signaling state structures. To the best of our knowledge, this is the first time an all-atom reconstruction from a coarse grained model has been performed in a relatively unexplored region of molecular configuration space. We compare our signaling state prediction with previous computational and more recent experimental results, and the comparison is favorable, which validates the method presented. This approach provides additional insight to understand the PYP photo cycle, and can be applied to other systems for which more direct methods are impractical.

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Section: Introductionmentioning

confidence: 99%

Multiscale Approach to the Determination of the Photoactive Yellow Protein Signaling State Ensemble

et al. 2014

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“…A previous study also reported the fusion of another coloured protein, photoactive yellow protein (or its miniaturized version), to a target protein. In this case, the addition of a precursor of the chromophore to the coexpressed photoactive yellow protein causes a yellow colour to appear; this colour development not only allows target protein expression to be monitored through visual inspection within a few seconds, but also enables protein concentration and purity to be quantified using a spectrometer within a few minutes [ 111 ].…”

Section: Expression Vectors For High-throughput Protein Expressionmentioning

confidence: 99%

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

Jia¹,

Jeon²

2016

Open Biol.

249

160

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The ease of genetic manipulation, low cost, rapid growth and number of previous studies have made Escherichia coli one of the most widely used microorganism species for producing recombinant proteins. In this post-genomic era, challenges remain to rapidly express and purify large numbers of proteins for academic and commercial purposes in a high-throughput manner. In this review, we describe several state-of-the-art approaches that are suitable for the cloning, expression and purification, conducted in parallel, of numerous molecules, and we discuss recent progress related to soluble protein expression, mRNA folding, fusion tags, post-translational modification and production of membrane proteins. Moreover, we address the ongoing efforts to overcome various challenges faced in protein expression in E. coli, which could lead to an improvement of the current system from trial and error to a predictable and rational design.

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“…Numerous attempts have been previously made to develop a simple and rapid screening method. For example, in a study, the protein expression was monitored by the fusion of photoactive yellow protein to the target protein and the protein concentration and purity were quantified employing a spectrometer within a few minutes [ 18 ]. The method, however, needs the addition of a precursor to develop the color that imposes more cost to the system.…”

Section: Discussionmentioning

confidence: 99%

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

et al. 2021

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Background 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). Results 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. Conclusion 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.

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High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Cited by 14 publications

References 66 publications

Multiscale Approach to the Determination of the Photoactive Yellow Protein Signaling State Ensemble

Multiscale Approach to the Determination of the Photoactive Yellow Protein Signaling State Ensemble

High-throughput recombinant protein expression in Escherichia coli : current status and future perspectives

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system

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