PubMed · 14970554
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Iris West, Owen Goldring. 2004. Lectin affinity chromatography.. https://doi.org/10.1385/1-59259-655-x%3A159
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The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.
In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG/salt system. The presence of both types of residues in the tag increased the partitioning greatly. Particularly, four engineered tags (H6, FH6, WH6, and YH6) containing a hexa-histidine sequence as well as different hydrophobic residues, all increased partitioning more than twice, reaching K values around 20, as compared to another construct (His6-GFP) containing an isolated hexa-histidine sequence. YH6, also proved be beneficial for protein expression.
NADP-malic enzymes (NADP-ME) are isozymes in plants. To clarify the diversity and function of NADP-ME isozymes in rice, we produced two active GST-fused NADP-ME proteins, NADP-ME2 and NADP-ME3 in Escherichia coli, and the fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column. After enzymatic cleavage of the GST tag, final yields were 1.4 mg/g wet cell weight (wcw) for NADP-ME2 and 3.5 mg/g wcw for NADP-ME3, respectively, and the molecular weights of NADP-ME2 and NADP-ME3 were about 65 and 62 kDa, respectively. The optimum pH is 7.3 for NADP-ME2 and 7.7 for NADP-ME3. The Km values for malate of NADP-ME2 and NADP-ME3 were 2.6 and 3.1 mM, whereas the Km values for NADP were 79 and 93 microM, respectively. The Kcat values of NADP-ME2 and NADP-ME3 for malate were about 91.7 and 96.7 s-1, respectively, and the Kcat values for NADP about 88.3 and 98.3 s-1, respectively. These results suggest that the two rice isozymes of NADP-ME in vitro have similar kinetic parameter.