In addition, FFF enables flexible separation of inhomogeneous samples and collection of fractions for further analysis off-line. As an example of the utility of this approach, FFF / LS analysis of cathepsin B that was refolded in the presence of NDSB 201 pH 8 revealed the presence of a LS and DRI peak corresponding to a monodisperse species of ϳ 46 kDa, close to the predicted MW of the cathepsin B monomer (37.3 kDa). The same analysis performed on a refolded cathepsin B sample that produced good solubility but no activity showed poor refolding recovery. Refolding of cathepsin B from two different inclusion body preparations led to the same pattern of activity, indicating good reproducibility of the method. We are applying this method to refold insoluble T. maritima proteins and save failed targets in the JCSG framework.
To maintain the integrity of the pipeline structure, protein purity, quality, and identity are evaluated at various stages of the pipeline. After metal affinity purification, SDS-PAGE is used to assess the purity of the sample prior to secondary purification. The images of these gels are cataloged in an Access database for future reference. Relevant bands from these gels are excised and subjected to tryptic mass spectrometry to confirm the identity of the expected protein. Tryptic MS is also performed on proteins emerging from secondary purification to confirm that the integrity of the sample has been preserved through the above steps. In addition to evaluating the success of refolding, FFF / LS can also be used to determine the oligomeric status of protein samples. In order to maximize protein structure production through process optimization, the JCSG employs a 2-level strategy for prioritizing targets .
At the first level, target proteins are rapidly assessed for their ability to express themselves in soluble form and their propensity to crystallize. For T. maritima, level 1 proteome screening focused on processing as many of the T. maritima ORFs predicted in 1877 as possible through the process of cloning, expression, purification, and crystallization without extensive optimization of methods or purity. of the sample. Samples that were at least 90% pure as estimated by SDS-PAGE analysis were immediately prepared in crystal assays against 480 commercially available dispersed matrix conditions at 20 ° C. This effort resulted in the processing of 1376 unique proteins , with 539 unique proteins (ϳ 29% of the T. maritima proteome) prepared for crystallization assays. Of these, 465 (86%) produced crystal impacts, indicating that they could probably be optimized to provide diffraction quality crystals for structure determination.
This set of proteins had representatives of all protein classes in the T. maritima proteome (Table 3) and serves as an unbiased study of the targeting ability of the proteome for the determination of structure through our pipeline. The level 2 effort is focused on producing high-quality crystals suitable for structure determination. At this stage, the targets that crystallized on the level 1 proteome screen are prioritized according to the probability that they contain novel folds or novel structural features. Targets of more than 820 residues and small of 60 residues and potential membrane proteins are excluded at this stage. These targets are then reprocessed through the expression and purification pipeline, this time producing selenomethionine-labeled protein and using secondary purification steps (Table 4).
The Tier 2 effort currently consists of 269 objectives. Of these, 104 targets are part of an effort to develop molecular replacement methods using a partial model, 132 require production of selenomethionine-labeled protein, and 33 are being resolved by NMR methods. Currently, 105 of the selenomethionine targets have been expressed and crystallization conditions have been identified for 103 of them. To date, this 2-tier approach has resulted in 55 resolved structures (5 pending), 6 of which have been identified as new folds.