Proteome-scale purification of human proteins from bacteria

Abstract

The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequence-verified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.

Figure 1

Expression of 32 test set proteins fused to four different purification tags. The 32 genes in the test set were transferred into each of the four expression vectors, transformed into BL21 cells, and cultured and induced as described. 10 μl (≈1%) of each culture was lysed directly in Laemmli buffer and analyzed by Western blotting using antibodies against the peptide tags as indicated. Bands of the correct size are indicated by a dot on the right side of the band.

Figure 2

Summary of all test set protein purifications: All 128 proteins were purified by using the respective affinity tag, and total yield and purity of the purified proteins were analyzed by GelCode staining and image analysis. Losses were characterized by Western blot analysis of five key fractions. Yield: red, <300 ng; light blue, 300 ng to 1 μg; dark blue, >1 μg. Purity: red, <10% purity or no detectable band; light blue, 10–30% purity; dark blue, >30% purity. Losses: red, protein degraded in vivo; orange, >60% of lost protein in the flow-through; dark yellow, losses evenly distributed between flow-through and matrix; bright yellow, >60% of lost protein was found on the matrix.

Figure 3

Test set proteins as purified with the GST tag (A) and the MBP tag (B). Bands of the correct size are indicated by a dot on the right side of the band. Fifteen percent of the total eluate was loaded on a 4–20% gradient SDS-gel and stained with GelCode Coomassie blue reagent.

Figure 4

GST- and MBP-tagged proteins are active. Autoradiograms of ³²P incorporation. (A) Equal amounts of bacterially purified GST- and MBP-tagged cyclin E were added to GST-cdk2 purified from insect cells in histone H1 kinase reactions. Both constructs activate cdk2 kinase activity. (B) p16^Ink4a specifically inhibits kinase activity. Equal volumes of 16 GST-tagged test set proteins were added to kinase reactions using cyclin D1/cdk4 purified from insect cells and C-terminal fragment of pRb as a substrate.

Figure 5

HT protein purifications. Multiple isolates of 336 different proteins were expressed and purified under denaturing conditions. Of these, 204 cDNAs gave rise to a Coomassie blue-stained band of the expected size. The corresponding proteins were expressed as GST fusions and purified under nondenaturing conditions. (A) Seventy-five different proteins successfully purified under denaturing conditions by using the His₆ tag. (B) The same 75 proteins fused to GST and purified under nondenaturing conditions. Fifteen percent of the eluate was loaded in all lanes.

Figure 6

Correlation of purification success to biological properties of the proteins. The success of protein purification under denaturing conditions was related to the molecular localization of the proteins in the mammalian cell (A) and to Pfam domains of the proteins (B).