Poor expression levels, solubilization, and stability are well-known challenges in the production of recombinant membrane proteins. Selecting the best expression conditions typically focuses only on expression levels, while protein stability characterization is carried out separately, sometimes after protein purification.
Andromeda increases the efficiency of CROs and Biopharma protein production teams by optimizing expression conditions and offering knowledge of expression levels and thermal stability from a single assay before protein purification.
Andromeda increases the efficiency of your protein production team
Screen more expression conditions faster in crude lysates
Routine workflows rely on more than one method to screen expression and extraction conditions — like SDS-PAGE and SEC or FSEC. Use Andromeda instead of these time-consuming and lower throughput methods. In a capillary format, analyze up to 48 crude lysates in just 11 minutes with little sample processing so you move on to scale-up and purification sooner.
Determine if your protein is highly expressed and properly folded in a single assay
To measure thermal stability, you either have to purify protein first or with crude lysates, you perform tedious, low throughput FSEC-TS. Andromeda easily identifies expression conditions that both yield sufficient and properly folded protein in crude lysates — because learning earlier if your protein is stable means not having to go back to screen more conditions.
Assess your protein’s binding activity by thermal shift assay before committing to purification
When producing a target protein for affinity screening studies, the expectation is you’ll deliver adequate amounts of structurally stable product. Andromeda quickly evaluates your protein’s thermal stability in the presence of ligands in crude lysates during small-scale expression.
Use Andromeda in your pharmaceutical protein production workflow
As a protein production team member, your job is to provide high-quality target proteins for biophysical characterization, affinity screening, and structural biology groups. Often, they put the quality of the proteins to the test — and when things don’t work as expected, your team is blamed.
Here’s what you can expect with Andromeda in your protein production workflow.
Save time by not having to repeat expression screening
If you know the stability of the protein early on, you purify the most stable target and save yourself the trouble of re-screening
Hand over high-quality proteins faster
Reduce the overall time it takes you to send purified proteins to the structural biology, biophysics, or screening teams by monitoring expression levels and thermal stability in one assay
Make informed decisions during low-scale expression
Evaluate not only expression levels but also stability early on, so you choose the right expression conditions before committing to purification
Make your protein service business stand out with Andromeda
Whether you’re a protein service provider or a CRO, your promise to your clients is to deliver the highest quality proteins on time and at a competitive price. But when it takes you multiple rounds of expression screening, scaling up and expensive purifications to optimize conditions, it’s hard to make good on those promises.
Here are some of the benefits Andromeda can bring to your protein services.
Lower your costs
Optimize expression conditions at the small-scale stage without scaleup or purification needed
Offer your clients shorter turnaround times
Reduce the time it takes to get to the purification stage by monitoring multiple expression conditions at once
Get in-depth knowledge before expensive scaling up or purification
Get information not only on expression levels but also stability
Make your protein services stand out
Be the preferred choice amongst other protein services providers
See if Andromeda can analyze lysates from your expression system
Instrument, consumables, and software designed to work in harmony
When it comes to software, AN.Control assay setup is simple and data interpretation is straightforward. Then when you need more advanced analysis options, get AN.Stability Analysis for instant stats and data analysis of merged replicates across multiple runs.
Choose this system if you want throughput flexibility. Measure from 1 up to 48 samples in individual capillaries.
Your best option if you prepare samples in 96-well plates. Lysates can be loaded into capillary chips directly from the wells.
Andromeda uses thermal unfolding to help you select expression conditions that ensure the stability of your protein
Andromeda measures the thermal unfolding of a protein in crude lysates by detecting changes in fluorescence — from either site-specific labeled His-tagged proteins or directly from GFP fusion proteins — during a thermal ramp. Interpreting the data is straightforward: a peak in the first derivative graphic means your protein is properly folded. Additionally, the inflection point at which the unfolding occurs (Ti) can be used to assess the stability of the protein. And since expression conditions can affect expression levels and stability, the combination of Ti shift and peak height tells you the relative abundance and whether the protein is properly folded or not.
Get your lysate ready in just one step – or no steps at all
In crude lysates, Andromeda detects fluorescence from your recombinant membrane protein to determine expression levels and stability.
For GFP fusion proteins, just load your lysate into the capillary and start measuring. For His-tag proteins, simply use the 1-step Andromeda His-tag labeling kit for site-specific and purification-free labeling. That’s it! Now you can start measuring.
Get information about expression levels, stability, and ligand binding
There is a lot to learn about your recombinant protein from Andromeda’s data — so you move forward to purification with candidates that are stable and more likely to be functional. Here’s an overview of what the data tells you.
Compare peak heights of lysates collected from different expression conditions — like growth media or temperature — to select those that result in high expression of folded recombinant protein.
Use shifts in the first derivative peaks to find out which conditions stabilize your protein.
Perform thermal shift assays to monitor the binding activity of your protein to a ligand.
Screen for the optimal E.coli strain
Unfolding profiles represent the average of at least three technical replicates. The shaded area indicates the standard deviation amongst all technical replicates.
When expressing a protein in an organism other than its native host, a variety of issues can arise. Genetically engineered E. coli strains with enhanced capabilities that overcome toxicity or reduced translational efficiency are available. In this experiment, three such strains were used to express His-tagged HRV3C.
Clarified lysates from three E.coli strains expressing His-tagged HRV3C were checked with Andromeda to determine which host resulted in the highest expression level of properly folded protein. First derivative traces show differences in the yield of properly folded protein amongst the strains and indicate that strain Rosetta2 (DE3) pLysS expresses the highest level of stable His-tagged HRV3C.
Read the technical note to see the rest of the expression screening, plus a comparison to SDS-PAGE.