Cradle is a Generative AI platform to design better proteins. But that’s only part of the equation—at some point, we must determine whether the proteins designed in silico perform the way our models predict they will. And of course, that means growing and purifying those proteins for downstream experimentation. 

At Cradle, we’re not just about modeling—we know that the strength and performance of our AI models is intimately linked with functional data we acquire in our in-house laboratory. We express, purify and test hundreds of proteins designed by our models every week, and we achieve this throughput with an optimized protocol that doesn’t require millions of dollars and can be adapted to be used with and without super fancy machines.

Imagine how your research programs might change, the questions you might be able to answer, if you could express, purify and test 384 proteins at once instead of just one or two in a week. We want your research to benefit from high throughput protein purification as much as our own R&D efforts have, which is why we’re sharing with you the exact protocol we use in our lab for a fast, high-throughput, and accessible protein purification workflow.

An uncommon solution to a common problem

Traditional protein purification workflows require researchers to address scale-up challenges both at the culture stage and the purification stage. The vast majority of protocols use LB based media to grow the protein-expressing microbes and liquid to purify protein from culture. But neither of these methods are easily adapted to high-throughput protocols, due to the demand for high starting culture volume and a slow, gradual purification process. Fast growing microbes tend to direct most of their metabolic energy into making more cells, other than producing the protein of interest. This means that the protein concentration in the cell will be low, and thus large amounts of culture will be required to obtain a sufficient amount of protein. 

Many organizations scale up using expensive, automated solutions purpose-built to address these challenges. But such solutions aren’t accessible to start-ups or academic research labs with smaller operating budgets and limited access to instrumentation or core facilities. As a start-up ourselves, we began to think outside of the box, figuring out how to take the traditional workflows and make them effective at minimal culture volumes, so that we would be able to grow our proteins in multi-well plates, instead of large culture flasks.

A new use for research tools

We aimed for outside the box idea and that’s exactly what we achieved: we’ve developed a protocol that can express high amounts of protein in relatively small volumes, and purify 384 proteins in only two hours using media and equipment that wasn’t designed with protein purification in mind—but perfectly addresses the culture and purification throughput challenges faced by researchers.

First, we addressed the culture problem. As mentioned, traditional approaches using LB media lead to low or inconsistent growth, measured through optical density (O.D.). We use the advanced, ready-to-use culture media produced by EnPresso® to achieve consistently high O.D.s (~30 in 1 mL culture in a 96-well format) from our cultures.The controlled carbon source release system of EnPresso forces cells to undergo linear growth as opposed to an exponential growth. Due to the controlled glucose release system, the specific substrate uptake rate for fast-growing cells is limited, allowing cells with a longer lag phase to catch up with them by the end of the incubation. Using this media together with our mid-expressing plasmid, we achieve 15-50 μg of pure, active, and natively folded protein from just 1 mL of culture.

As with any protein purification workflow, a single media isn’t one-size-fits-all, and we’ve had to engage in some optimization exercises to achieve similar levels with more difficult proteins, such as large protein complexes. Nevertheless, we achieve 100% pure protein on 80% of the proteins that include traditional enzymes, nanobodies, and polymerases we grow using the EnPresso® media. And perhaps even more importantly, the second half of our optimized protocol — the purification stage — works 100% of the time.

We purify our proteins from EnPresso® cultures using the BlueCatBio Blue® Washer XL —a centrifugation-based plate washer with a magnetic plate attached for processing samples containing magnetic beads. This instrument has an extremely small footprint and can purify 384 proteins in only two hours. While the Blue®Washer is conventionally used for reducing tip-use in plate assays, it has proven to be a perfect fit for protein purification as well.

This protocol is critically important to us at Cradle. As explained by Michelle, “Having this high-throughput setup and making it as robust as possible means that the Bioengineering team can provide highest quality data to the machine learning models with a quick turnaround time.

So what if you don't have a BlueWasher? Well, we have tested and confirmed that one can achieve an equally high purified protein yield without the need for one. For initial and lower cadence testing, one can use a high quality 96 well magnetic plate such as the Alpaqua magnetic plate and multi-channel pipettes to mimic Blue® Washer’s protocol. 

Accessible protein purification

Although we developed this protocol to advance our own models, we believe the importance of this protocol goes beyond what occurs inside the walls of our own lab.

“It's really impressive to be able to see that not only can these machine learning algorithms and models churn out all these really cool candidate proteins, but we can actually validate which ones work well and which ones don't in our lab. And instead of just picking the top five or the top 10, we can test hundreds all at once in a relatively small timeframe, which enables the one-after-the-other iterative cycles of protein engineering to really take off,” explains Michelle.

And that’s exactly why we want to share our protocol: not just to demonstrate that protein engineering can happen quickly using our approach, but to provide a battle-tested guide for other scientists.

“Something as simple as providing this workflow makes it more accessible. We’re trying to change the mentality because a lot of companies think that getting fancy equipment will solve all of their problems, but it’s not really like that. Science is not as complicated as we often think and we don’t necessarily need millions of dollars to make it work,” explains Jinel.

Michelle and Jinel also think it’s important that researchers use what we’ve achieved at Cradle to inspire their own think-outside-the-box innovation.

“It’s useful that we can provide this protocol to our clients and say ‘this is how we test our models in house, and maybe you can implement some of these assays and protocols directly into your workflows,’ but even if they don’t have access to or can’t buy fancy equipments, they can start thinking about creative ways to make their protocols work on a smaller, more high-throughput scale,” says Jinel.

Protocol at-a-glance

We hope we’ve shown you that high-throughput protein purification doesn’t have to be complicated. You can achieve high-purity, rapid protein purification using a fairly simple protocol.

Below are the basic steps we follow in our own lab to purify and validate hundreds of proteins designed by our ML models.

Cell Growth with EnPresso®

To achieve optimal cell growth, simply inoculate your colonies or glycerol stocks into seed cultures in 96 well plates. After 5-8 hours, back-dilute your seed cultures into prepared EnPresso expression culture media, incubate for 15-18 hours. Now, boost protein expression by adding EnPresso's Booster media, and incubate for 24 hours. Proteins are now ready for purification!.

Cell Lysis and Purification with Blue®Washer

To prepare cells for protein purification, spin your plates to pellet the cultures and lyse your cells with Fastbreak lysis buffer. Add 0.5M NaCl to your lysate to improve binding to MagneHis™ Ni-Particles.

Next, add Magnehis beads and lysate to a Blue®Washer plate. Run the purification protocol, remove your plate from the Blue® Washer, separate the magnetic beads from the elute, and transfer purified protein to a fresh 96 well plate. If you don't have a BlueWasher, a protocol for purification using only a magnetic plate and a multichannel pipette is also provided below.

Purified proteins are ready for downstream processing such as buffer exchange, glycerol stocking, expression analysis and assays.. A variety of methodologies can be used to measure protein concentration and purity, including A280, BCA, Bradford assay, Lab chip, or SDS-PAGE.

To view our full protocol for high throughput protein purification using the Blue®Washer and EnPresso® media tablets, click here.