PubMed · 7765549
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I Y Galaev, B Mattiasson. 1994. Shielding affinity chromatography.. https://doi.org/10.1038/nbt1194-1086
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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.
A two-step chromatographic procedure for purification of basic fibroblast growth factor (bFGF) from high-cell-density cultures of recombinant E. coli is described. Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for purification of bFGF from the soluble cell fraction. A one-step affinity chromatographic procedure resulted in very pure bFGF. However, this one-step affinity isolation of bFGF caused the loss of around 60% of the recombinant protein. A combination of ion-exchange chromatography with heparin-Sepharose affinity chromatography was favored for bFGF purification. A first cation-exchange chromatographic step resulted in a solution of bFGF with a purity of around 70%. The weak cation exchanger CM Sepharose C50 was preferred in comparison to the strong cation exchanger S-Sepharose because of the higher recovery of bFGF. With the ion-exchange chromatographic step prior to the heparin-Sepharose affinity chromatography, the total yield of recovery of bFGF increased to 56% compared to 40% using the one-step purification procedure with heparin-Sepharose. To characterize the equilibrium parameters of adsorption, batch experiments for the calculation of maximum capacities and dissociation constants for CM-Sepharose C50 and heparin-Sepharose were carried out. The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction according to the Langmuir model of adsorption. The adsorption of bFGF to heparin-Sepharose was described by a double Langmuir approach of two independent binding sites with different maximum capacities and dissociation constants. The purified bFGF showed a high biological activity and circular dichroic spectra of a proper folded molecule. The analysis of the N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first amino acid methionine. In addition, half of the bFGF molecules lacked the second amino acid alanine.
Many of the most widely employed operations in molecular biology hinge upon the use of single-stranded DNA as a probe or template. Here we report a straightforward method by which to produce long single-stranded DNA molecules using the polymerase chain reaction (PCR) in combination with immobilized metal affinity chromatography (IMAC). We demonstrate that a tag consisting of six successive 6-histaminylpurine (H) residues (H6-tag) endows a DNA strand with selective retentivity onto a Ni2+-NTA-agarose chromatography matrix. The H6-tagged strand can then be eluted from the resin using 200 mM imidazole. Quantitative phosphorimaging analysis revealed that the PCR/IMAC procedure typically yields unmodified strands comprising >90% of the unbound DNA and H6-tagged strands comprising >95% of the bound fractions. DNA strands generated in this manner are shown to be excellent substrates for template-directed polymerization. The chemistry reported herein should facilitate a wide variety of operations in molecular biology, including automated DNA sequencing, hybridization screening of DNA libraries, assembly of gene cassettes, run-off transcription, site-directed mutagenesis and footprinting of protein-DNA complexes by template-directed interference footprinting.