Thread by @RusanLab

A solubility screen to identify centrosome protein complexes

Our collaboration with @GregoryCRogers1 is online @JCellBiol and I wanted to highlight one aspect that is somewhat buried, but took my lab nearly 2 years to complete

the pExpress-Dual screen

rupress.org/jcb/article/21…

Frustrated by years of failed centrosome protein purifications for biochemical assays and crystallography, we realized that - proteins need their partners to be HAPPY - . So Sarah Speed, a postbac, designed a screen to isolation soluble centrosome protein complexes

Each gene was amplified as FL and/or sub-fragments with two different pairs of primers for a total of 106 PCR products + 2 PCR products that complete the plasmid. One MEGA-GIBSON REACTION was conducted to randomly assemble pExpress-Dual plasmids.

Following transformation, random selection of 1000 colonies, and culture growth in deep 96-well format, we proceeded through a two step screen. The pipeline included tandem IPs for His-(position 1) and GFP-(position 2), removing false positive and duplicates, then scaling up.

The result of the screen was the isolation 21 centrosome protein complexes that were soluble and stable enough to survive the protocol. While our paper only highlights 6 of these interactions, it is the list of 21 that are very promising for biochemical and structural studies.

The 6 PPIs we highlight are ones that we reciprocally IP-ed
- Three interactions previously studies by us and other (Plk4-Plk, Plk4-Asl, Ana2-Ana2).
- One unexplored interaction (Cnn-Ana2)
- Two interactions that are the main focus of this paper (Plk4-Sas4, Ana2-Sas4)

There you have it-necessity is the mother of invention. A failure to isolate well behaved individual centrosome proteins forced us to isolate complexes. I am proud that this was designed and executed by my NIH postbac Sarah Speed (thumbs up) with help from tech Carey Fagerstrom