Table 1.
Complete additive and buffer list at final concentrations.
Figure 1.
Flowchart of the additive screen in the context of high-throughput structural genomics.
The target open reading frame is cloned into SSGCID vectors then expression testing is performed [25]. If the protein is soluble it is entered into our standard purification pipeline [28]. High priority insoluble proteins enter into additive screening. One two-liter culture is grown to obtain pelleted E. coli expressing the recombinant proteins for the screens, and then the 144 condition primary screen is conducted. All conditions that show an increase in solubility are subjected to a secondary screen for confirmation. Successful secondary screen proteins are then purified with additives and those that are >95% pure and yield >5 mg enter into crystal trials. In the event that the secondary screen fails, the results from the primary screen may be used to guide the creation of new additive and buffer combinations for custom screening.
Table 2.
Screening step success rate.
Table 3.
Individual protein screening and scale-up results.
Table 4.
Top additives results.
Figure 2.
SDS-PAGE gels from the screen of T. gondii Hsp20.
(A) Presented is one of the six SDS-PAGE gels from the primary screening experiments of Hsp20 from Toxoplasma gondii. Six screening gels are performed per protein to screen all 144 unique cell lysis conditions, 24 conditions per gel. Each gel is run with the protein-lysed apo (without additives) on the far left side of the gel. “Std. Total” is total cell lysate, lysed without additives. “Std. Soluble” is the soluble fraction of the non-additive lysed cells. The lanes between ‘A1’ and ‘B12’ are the 24 conditions screened on this gel. The expected molecular weight of the recombinant protein, Toxoplasma gondii Hsp20, is indicated with the arrow. The conditions that appeared to increase solubility and were subsequently re-screened are LDAO (A2), L-Arginine (B2), L-Proline (B3), Glycine Betaine (B4), Mannitol (B7), Trehalose (B8), NDSB 195 (B11), and Trimethylamine N-Oxide (B12). (B) SDS-PAGE gel image shows the individual secondary screen for the Toxoplasma gondii Hsp20 from figure 1A. The Toxoplasma Hsp20 protein has an ‘Apo’ control that was lysed without any additives present. Additive conditions are in the lane to the right of each Apo. Lanes marked ‘T’ are the total cell lysate, lanes marked ‘S’ are the soluble fraction for each condition. The overexpressed protein band at ∼28 kDa is the protein of interest. Conditions B8 (Trehalose) and B11 (NDSB 195) proved to be the best in solubilizing the protein, where close to 100% of the protein was present in the soluble fraction when compared to the total cell lysate, as opposed to conditions B2–B7 where there is a clear distinction between the relative amount of recombinant protein present in the soluble fraction compared to the total fraction.
Figure 3.
The top performing additive molecular structures.
Presented are the molecular structures of the top performing additives. The number of proteins soluble with each additive is presented in table 4.
Figure 4.
SDS-PAGE of the purification of Coccidioides posadasii Proline-Rich antigen 2 (Prp2).
SDS-PAGE gels from Prp2, which was purified in the presence of 100 mM potassium citrate. The resulting protein resulted in diffraction quality crystals. On the left are samples from the metal affinity purification step. Lanes are as follows: “M” molecular weight standards with the corresponding weights in kDa indicated; “T” total cell lysate; “S” soluble cell lysate after centrifugation; “FT” flow through from the affinity purification column; “E” eluate from the affinity column. The band corresponding to Prp2 is marked the arrow “P”, the arrow “L” is lysozyme added during lysis. Select size exclusion chromatography fractions were analyzed via SDS page, the fraction deemed the purest concentrated for crystal trials are boxed. This protein formed diffracting crystals but the structure has not yet been solved.
Figure 5.
Proposed mechanism for rescue of recombinant protein solubility.
We hypothesize that up to 80% of the seemingly insoluble recombinant proteins are in a partially folded state and reside in the E. coli cytosol. If lysed in a non-ideal buffer, the proteins unfold, resulting in aggregates of insoluble protein. When the sample is centrifuged to separate the soluble fraction, the protein aggregates are present in the insoluble cell pellet. If the additives are present during cell lysis, they can either stabilize the proteins from partially unfolding, preventing protein-protein interactions, or aid as chemical chaperones, leading to the properly folded and non-aggregated state. When centrifuged there are minimal protein aggregates and the recombinant protein remains in the soluble fraction.