A High-Throughput Multiwell-Plate based Approach for the combined Expression, Export and Assay of Recombinant Proteins
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (Arcadia Science)
Abstract
High-throughput screening (HTS) of proteins has a wide range of applications across the biology, biotechnology, and medicine disciplines. These include yield optimisation, drug or biomarker discovery, and protein engineering, among others. Factors that need to be considered in designing high throughput protein expression and screening methods, be that for expression, activity, stability, or binding as says, include the required yield, reproducibility, solubility, stability, purity and activity of the protein. Thus, larger culture volumes and time-consuming manual protein extraction and purification steps are normally required to produce sufficient quantity of protein of appropriate purity. This limits the type of assay, and number of protein variants that can be simultaneously tested in an experiment. Here we describe a HTS protocol that allows the overnight expression, export and assay of recombinant proteins from E. coli cells in the same multi-well plate tube. The protocol uses a recently described Vesicle Nucleating peptide (VNp) technology that promotes high yield vesicular export of functional proteins from E. coli into the culture media. The resulting protein is of sufficient purity and yield that in can be used directly in plate-based enzymatic assays without additional purification. This simple single plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications.
Article activity feed
-
This simple single plate protocol allows itself to a wide range of high-throughput research and development screening applications, ranging from streamlining protein production and identification of activity enhancing mutations, to ligand screening for basic research, biotechnological and drug discovery applications.
This is a really interesting method using a peptide tag to target proteins to extracellular vesicles for ease of isolation in E. coli! I can think of lots of benefits and applications!
-
As an illustration, we have developed a multiwell format in vitro assay that allows researchers to measure the activity of in-plate expressed and exported VNp-uricase protein (Figure 3), by following changes in 293 nm absorbance to monitor enzyme dependent breakdown of uric acid
I'm guessing that you measured this in your initial paper, but might be worth mentioning here as well. Have you shown that the VNp tag doesn't affect enzyme activity, stability, folding?
-
5 min spin
At what speed?
-
The VNp system has the additional benefits of not only allowing expression of more challenging proteins, but also expressing proteins at higher yields than standard methods will normally allow
I'm sure you talk about this in your previous paper, but it would be interesting to know the range of proteins that you've tested with this method since you talk about the system being useful for more challenging proteins.
-
Schematic of vesicle cleavage and plate-based assay.
So you isolated the vesicles, lysed them, and then went straight to the assay, or did you do any sort of affinity purification or centrifugation or anything here?
-
The VNp tag facilitates the export of recombinant proteins into extracellular membrane-bound vesicles, creating a microenvironment that enhances the solubility and stability of challenging proteins
Very cool!
-
However current methodologies require extraction and purification of protein from cells requires that often require manual input to the process, and thus roadblocks to the HTS process.
A very minor thing, but this sentence seems a bit odd.
-
