USP Zf-UBD Crystallography Pipeline Pt 2.

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

 

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

 

 

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