In a previous post, I described the importance of solving the structure of other USPs that have a Zf-UBD for the rational design of selective compounds. The last post described the design, cloning, E.coli expression, growth and purification of some of the USP Zf-UBD constructs. As a continuation in my quest to try and crystallize the other USP Zf-UBDs I’ve finished purifying and setting up crystallization screens of the ‘TOC013’ constructs in an attempt to crystallize these protein samples for subsequent structure determination by X-ray crystallography. You can see details on Zenodo. I previously described getting crystals of USP33 that did not diffract and USP20 microcrystals that were too small to mount. I tried ‘seeding’ these initial crystal conditions in the hopes of getting larger crystals that diffract. Micro-seeding is the process of adding a dilute solution of micro-crystals to a crystallizing solution to nucleate more crystals. This helps in controlling size and reproducibility.
So far, I have not had any luck with seeding but it may just be a matter of time- some proteins take longer to crystallize than others. I’ll have to keep an eye on my plates. Hopefully I’ll have some luck in the New Year and come back to crystals after the holidays! The USP Zf-UBD crystallization pipeline to date is summarized in Table 1.
Table 1. Summary of USP Zf-UBD Crystallization
| Clone | Target | AA | Vector | Media | Amount Grown (L) | Purification Yield (mg) | Crystal Plate Setup (Y/N) | Crystals? (Y/N) | Diffraction (Y/N) |
| TOC013E09 | USP44 | 27-110 | pET28-MHL | M9 | 12 | No/low expression | Y | N | |
| TOC013F02 | USP49 | 15-105 | pET28-MHL | M9 | 12 | 0.25 mg | Y | N | |
| TOC013D10 | USP33 | 37-124 | pET28-MHL | M9 | 6 | 15.7 mg | Y | Y
-seeding:100 nL of 1:1000 and 1:10000 SM of 1 µL drops of RWD01, RWE01, SGCF03 small microcrystals (SM concentration too high?) |
N |
| TOC013D11 | USP33 | 38-124 | pET28-MHL | M9 | 6 | 0.35 mg | Y | N | |
| TOC013E02 | USP39 | 97-197 | pET28-MHL | M9 | 6 | 1.6 mg | Y | N | |
| TOC013E05 | USP39 | 97-205 | pET28-MHL | M9 | 6 | 1.1 mg | Y | N | |
| TOC013C02 | USP13 | 188-292 | pET28-MHL | M9 | 6 | Insoluble | |||
| TOC013C03 | USP13 | 208-292 | pET28-MHL | M9 | 6 | Insoluble | |||
| TOC013C04 | USP13 | 208-308 | pET28-MHL | TB | 6 | 2.4 mg | Y | N | |
| TOC013B11 | USP03 | 10-152 | pET28-MHL | TB | 6 | 2 mg | Y | N | |
| TOC013C08 | USP16 | 26-146 | pET28-MHL | M9 | 6 | 0.7 mg | Y | N | |
| TOC013C09 | USP16 | 32-146 | pET28-MHL | TB | 6 | 0.8 mg | Y | N | |
| TOC013C11 | USP20 | 1-100 | pET28-MHL | M9 | 6 | 4.8 mg | Y | N | |
| TOC013D03 | USP20 | 1-110 | pET28-MHL | M9 | 6 | 13.2 mg | Y | Y: small microcrystals SGCC08
optimizing: 1) different protein concentrations (1, 2, 3, 4 mg/mL); no crystals 2) seeding with SGCC08; no crystals
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| TOC013D04 | USP20 | 8-110 | pET28-MHL | M9 | 6 | 7.2 mg | Y | N | |
| TOC013-D07 | USP22 | 43-142 | pET28-MHL | M9 | 6 | No/low expression | N | N | |
| TOC013D08 | USP22 | 49-142 | pET28-MHL | M9 | 6 | No/low expression | Y | N | |
| Constructs previously available in SGC database that have high expression: | |||||||||
| SDC093D04 | USP13 | 183-307 | pET28-MHL | TB | 6 | 26.3 mg | |||
| SDC093D05 | USP13 | 187-301 | pET28-MHL | TB | 6 | ||||
| SDC024F07 | USP16 | 34-185 | pET28-LIC | TB | 6 | ||||
| SDC024F09 | USP16 | 16-185 | pET28-LIC | TB | 6 | ||||
| SDC024F10 | USP16 | 8-185 | pET28-LIC | TB | 6 | ||||
| SDC036B06 | USP20 | 1-141 | pET28-LIC | TB | 6 | ||||
| SDC042G05 | USP33 | 7-139 | pET28-LIC | TB | 6 | ||||
| SDC042G06 | USP33 | 7-167 | pET28-LIC | TB | 6 | ||||
| SDC042G07 | USP33 | 7-183 | pET28-LIC | TB | 6 | ||||
| SDC168E10 | USP44 | 1-167 | pET28-MHL | TB | 6 | ||||
| SDC231F11 | USP51 | 160-350 | pET28-LIC | TB | 6 | ||||
| SDC231-G03 | USP51 | 176-350 | pET28-LIC | TB | 6 | ||||