Single-step protein purification by back flush in ion exchange chromatography

Chern, Ming-Kai; Shiah, Wei-Jyh; Chen, Jyun-Jie; Tsai, Tzung-You; Lin, Hwei-Jen; Liu, Chien-Wei

doi:10.1016/j.ab.2009.05.045

Cited by 8 publications

(8 citation statements)

References 6 publications

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“…S1 in the supplemental material). Bud23 had previously been shown to accumulate in inclusion bodies when expressed alone in E. coli (2), similarly to Mtq2 and Trm9 (15,36). Strikingly, we observed that the coexpression of Trm112 and His 6 -Bud23 increased the solubility of Bud23 by a factor of ϳ5 (Fig.…”

Section: Resultssupporting

confidence: 50%

Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575

Figaro

Wacheul

Schillewaert

et al. 2012

Molecular and Cellular Biology

122

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Posttranscriptional and posttranslational modification of macromolecules is known to fine-tune their functions. Trm112 is unique, acting as an activator of both tRNA and protein methyltransferases. Here we report that in Saccharomyces cerevisiae, Trm112 is required for efficient ribosome synthesis and progression through mitosis. Trm112 copurifies with pre-rRNAs and with multiple ribosome synthesis trans-acting factors, including the 18S rRNA methyltransferase Bud23. Consistent with the known mechanisms of activation of methyltransferases by Trm112, we found that Trm112 interacts directly with Bud23 in vitro and that it is required for its stability in vivo. Consequently, trm112⌬ cells are deficient for Bud23-mediated 18S rRNA methylation at position G1575 and for small ribosome subunit formation. Bud23 failure to bind nascent preribosomes activates a nucleolar surveillance pathway involving the TRAMP complexes, leading to preribosome degradation. Trm112 is thus active in rRNA, tRNA, and translation factor modification, ideally placing it at the interface between ribosome synthesis and function.A ctively growing budding yeast cells produce an average of 33 ribosomes per second, which is considerable (61). Besides the synthesis of its constituents, i.e., 79 ribosomal proteins and 4 rRNAs, ribogenesis involves a large number of so-called transacting factors, including proteins and small nucleolar RNAs (13, 56). These interact only transiently with preribosomes and are required for pre-rRNA processing (i.e., cleavages), pre-rRNA modification, and preribosome assembly and transport.Ribosome synthesis is initiated in the nucleolus, a highly dynamic specialized subcompartment of the nucleus organized around clusters of actively transcribed rRNA genes (60). There, RNA polymerase I produces a 35S/47S (in yeasts and humans, respectively) primary transcript which is processed through a complex succession of endo-and exoribonucleolytic cleavages into three of the four mature rRNAs, the 18S, 5.8S, and 25S/28S rRNAs. The fourth rRNA, 5S, is independently transcribed by RNA polymerase III. Ribosome maturation starts cotranscriptionally in the nucleolus, progresses in the nucleoplasm until preribosomes reach the nuclear pore complex, and is finalized in the cytoplasm. Cytoplasmic maturation steps consisting of pre-rRNA processing and structural reorganization result in the assembly of prominent ribosomal structures, such as the "beak" of the small subunit (SSU) and the "stalk" of the large subunit, and are a prerequisite to the acquisition of functionality (43).It is not clear how the various facets of ribosome synthesis are integrated with ribosome function. However, there is growing evidence that such coordination exists. For the small subunit, recent cryoelectron microscopy (cryo-EM) maps of late pre-40S subunits indicate that the binding of trans-acting factors literally mask functional sites, preventing premature translation initiation (57). For the large subunit, trans-acting factors that resemble ribosomal proteins (suc...

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Section: Resultssupporting

confidence: 50%

Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575

Figaro

Wacheul

Schillewaert

et al. 2012

Molecular and Cellular Biology

122

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“…For example, reversing the flow direction (“back flash”) or “codon optimization” in combination with “strikingly high isoelectric point” helped purify 1 or 2 proteins in a single-step [3, 6]. However, none of those studies provided adequate rationale or attempted to formulate to establish a unified method for protein purification.…”

Section: Discussionmentioning

confidence: 99%

“…Commonly used cation-exchange resins contain sulfate derivatives (S-resins), whereas the anion-exchangers (CM resins) have carboxylate derived ions. However, it rarely provides single step purification due to lack of specificity [3]. …”

Section: Introductory Statementmentioning

confidence: 99%

A unified method for purification of basic proteins

Adhikari

Manthena

Sajwan

et al. 2010

Analytical Biochemistry

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Protein purification is still very empirical, and a unified method to purify proteins without an affinity tag is not available yet. In the post-genomic era, functional genomics, however, strongly demands such a method. In this paper we have formulated a unique method that can be applied to purify any recombinant basic protein from E. coli. Here, we have found that if the pH of the buffer is merely one pH unit below the isoelectric point (pI) of the recombinant proteins, most of the latter bind to the column. This result supports the Henderson-Hasselbalch principle. Considering that E. coli proteins are mostly acidic, and based on the pI determined theoretically, apparently all recombinant basic proteins (at least pI-1≥6.94) may be purified from E.coli in a single-step using a cation-exchanger resin, SP-sepharose, and a selected buffer pH depending on the pI of the recombinant protein. Approximately, two-fifths of human proteome, including many if not all nucleic acid interacting proteins, have a pI of 7.94 or higher; virtually all these 12,000 proteins may be purified using this method in a single-step.

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“…In Ft-IEC, depending on the buffer with a narrow range pH lower or higher than the pI of IgG, the positively or negatively charged IgG and trace contaminants can be recovered in the flowthrough and washing effluents, keeping the negatively or positively charged contaminants remain adsorbed on anion exchange resins or cation exchange resins. Similarly, several recombinant proteins, including aldehyde dehydrogenases and one methyltransferase, were purified by Ft-IEC with Q Sepharose Fast Flow [11]. In addition to pH, salt concentration of the buffer strengthens the detachment of IgG by influencing on the electrostatic interactions between proteins and resins [12].…”

Section: Introductionmentioning

confidence: 99%

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

et al. 2011

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A one-step purification process using flowthrough mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min -1 . Ft-IEC using TrisHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris-HCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.

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Single-step protein purification by back flush in ion exchange chromatography

Cited by 8 publications

References 6 publications

Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575

Trm112 Is Required for Bud23-Mediated Methylation of the 18S rRNA at Position G1575

A unified method for purification of basic proteins

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

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