[Kasia Baranowski]

Hello!
I would love to hear how you all quantify soluble protein expression when you have hundreds or thousands of constructs to test.

What is your high throughput method of choice?

SDS-PAGE? Bradford? BCA? Mass spec? HiBit? SEC HPLC? fluorescence?
Any methods that utilize pools of cells?

I am designing a project for gathering large amounts of protein expression data and am looking for opinions on high throughput methods to scope.

thank you
-Kasia

[Rod Chalk]

Hi Kasia,

You may find this helpful

https://doi.org/10.3390/chromatography1040159

Kind regards,

Rod

[Kelvin Lau]

I’ve never done that scale.

In terms of day to day (in then 5-20s) and what I see people do (protein design) in medium throughout (multiple 96 well plates) SDS-PAGE is still the workhorse. It gives the required information, proxy for identity via band size and relative abundance. For most a gel from whole cell lysate is already enough yes/no to move forward with candidates. For soluble, they would prefer to do a 96 well plate based affinity.

BCA/Bradford IMO is not informative enough as it’s difficult to know what protein is contributing to the signal.

SEC-HPLC is laborious, requires machines, and if from lysate is most likely not conclusive if the peak corresponds to your protein. Also does not work for proteins that do not absorb. Also your interpretation will be skewed for proteins that are oligomeric. Cleaning the columns and speed will also be an issue.

Fluorescence? You have to be more specific.

The key is finding a method that is specific and can work through complex medium.

All of this is in context of Ecoli.

If it were say secreted mammalian cell culture my prefer to is BLI.

[Rob Meijers]

Hi Kasia,

we use fluorescent readout from a plate reader to screen mammalian protein expression in high-throughput (~80 constructs per plate plus a serial dilution on a control protein). It does require two epitope tags (to capture the secreted protein and to detect the level of material).

We have just published this in the second edition of David Hacker’s Methods and Protocols book on Recombinant Protein Expression in Mammalian Cells:

https://link.springer.com/book/10.1007/978-1-0716-3878-1?sap-outbound-id=4BA116D291A5E8F9720654A3E8447BE05CDF265B&utm_source=standard&utm_medium=email&utm_campaign=000_LAN36_0000019083_Book%20author%20congrats%20NEW&utm_content=EN_34155_20240701&mkt-key=42010A0D55461EDD8BFCFD1BA2177B95

Unfortunately, it is still behind a pay wall but should be open access soon.

Best regards,

Rob Meijers
rob.meijers@proteininnovation.org

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