Ion filtration chromatography: A powerful new technique for enzyme purification applied to E. coli alkaline phosphatase

Kirkegaard, Leslie H.; Johnson, Timothy J. A.; Bock, Robert M.

doi:10.1016/0003-2697(72)90492-7

Cited by 46 publications

(12 citation statements)

References 10 publications

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“…The harvested cells were resuspended by sonication in 50 mM phosphate buffer (pH 7.0) containing 300 mM NaCl. K-tag-AP was purified by affinity chromatography with a Talon metal affinity resin (ClonTech, Palo Alto, CA), followed by anion-exchange chromatography (Kirkegaard et al, 1972) using a DEAE CIM disk column (BIA Separations, Slovenia).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Takazawa

Kamiya

Ueda

et al. 2004

Biotech & Bioengineering

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Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Takazawa

Kamiya

Ueda

et al. 2004

Biotech & Bioengineering

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“…For this purpose purified nucleoli from livers of untreated animals were sonicated as described for nuclei and centrifuged for 1 h at 105000 x g. After adjustment of the (NH4)2S04 concentration to 80 mM, the supernatant was applied onto a DEAE-Sephadex column according to Kirkegaard et al [29]. RNA polymerase I eluted at the end of the wash of the column (Fig.…”

Section: Fig 3 Chromatography Of Nucleolar R N a Polymerase I On Deae-mentioning

confidence: 99%

Multiple Forms of DNA‐Dependent RNA Polymerase I from Immature Chick Liver

Bieri-Bonniot

Dierks-Ventling

1977

European Journal of Biochemistry

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1. We found that a single injection of 17P-estradiol into immature chicks resulted within 24 h in a 2-fold increase of the transcriptional capacity of liver nuclei which involved both endogenous RNA polymerase I and I1 activities. Similarly, RNA polymerase activity I of purified nucleoli was also doubled.2. During purification of RNA polymerase I from controls and treated chicks the difference in activity was almost completely lost.3. RNA polymerase I could be resolved into two forms, IA and IB, on CM-Sephadex. Form IA increased and form IB decreased after estrogen treatment while the sum of the two remained constant. Form IA sedimented more slowly than form IB in glycerol gradients and was predominant in purified nucleoli.4. These observations suggest that there may be an equilibrium between the two forms of RNA polymerase I which may change in order to produce the translational shift-up which occurs after estrogen treatment.Multiplicity of DNA-dependent RNA polymerase has been shown to exist in many eukaryotic systems [l -41. The three major classes can be distinguished on the basis of differences in chromatographic properties, structure, a-amanitin sensitivity, salt and cation requirements and intracellular localization, as recently reviewed by Chambon [5]. This multiplicity suggests that specific genes or groups of genes might be transcribed by different RNA polymerases. Sufficient data have now accumulated to show that the nucleolar RNA polymerase I (or A) insensitive to a-amanitin synthesizes ribosomal RNA [6,7]. RNA polymerase I1 (or B) synthesizes heterogenous DNA-like RNA [8] and more recently the role of enzyme I11 (or C) in the synthesis of 5-S ribosomal RNA and tRNA has become known [9]. The relative proportion of class I, I1 and 111 varies among different cell types and under different physiological or metabolic conditions [ 10,113. In addition, each of these classes of DNA-dependent RNA polymerases does not always consist of one particular enzyme : there exists multiple forms of enzymes having the same requirements and identical functions but different subunits [9, this may relate to the very complex and by no means resolved mechanism of regulation of transcription.Modification of transcription by hormonal action is by now an established means to study its possible regulating factors. 17P-Estradiol injected into immature chicks induces in the liver within hours the synthesis of vitellogenin, the precursor of egg yolk protein [15-371. In correlation with the production of vitellogenin, one could observe an increased synthesis of polysomal RNA [15,18], a large amount of vitellogenin messenger RNA [19] and a net synthesis of ribosomal RNA concomitant with an increase in the elongation factors of translation EF-1 and EF-2 [20]. Our main interest focuses on the observed shift-up of the translational machinery and the work presented here concerns the changes of RNA polymerase I after estrogen treatment of immature chicks in relation to the transcription of the ribosomal genes. We wish to report here th...

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“…Chromatographic systems in general that use any active combination of molecular sieve and adsorption processes are called sievorptive chromatography. Intervent dilution chromatography, as well as the recently defined ion filtration chromatography (2), are specific modes of the general process of sievorptive chromatography. Although ion filtration and intervent dilution chromatography are conceptually distinct, in practice both processes may occur within the same column, as happens when ion filtration chromatography is used to "desalt" an enzyme sample.…”

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confidence: 99%

Intervent Dilution Chromatography: Concept for Separation of Strongly Interacting Macromolecules

Kirkegaard

Agee

1973

Proc. Natl. Acad. Sci. U.S.A.

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Intervent dilution chromatography separates interacting macromolecules by subjecting them to a dynamic environment in which the association constant is continuously varied. The dynamic environment is produced by using the sieving properties of a gel to repeatedly propel the molecular complex across an intervent boundary. Behind the boundary, at high inteirvent concentrations, the complex dissociates;'ahead of the boundary, the component molecules are separated by adsorption processes. By selective adjustment of the intervent composition of the sample and the conditions of column equilibration, the process is adapted to a particular need.This report describes the chromiatographic' concept and shows how parameters are adjusted to obtain the desired separation. In this study a particularly difficult separation, i.e., the separation of ribosomal proteins from ribosomal RNA, is chosen to illustrate the power of the procedure.Separation of macromolecules that interact strongly with each other is one of the most persistent problems of biochemistry. Although chromatography has been used to measure the amount of association between molecules (1), current chromatographic procedures have found only limited use in separation of molecules that significantly interact with each other. This limitation results from the fact that the basic chromatographic process demands that molecules behave independently. It is sometimes possible to alter the chromatographic environment to permit the molecules to behave independently while retaining sufficient interaction with the stationary phase to cause separation; however, such changes usually reduce the interactions necesary for separation to a point where resolution becomes impossible.Intervent dilution chromatography is a combination of molecular sieve (gel filtration) and adsorption chromatography that effectively circumvents the stated limitation in classical chromatography. Because molecular sieve processes may be combined with adsorption chromatography in several ways to optimize for various needs, we suggest'some systematic nomenclature. Chromatographic systems in general that use any active combination of molecular sieve and adsorption processes are called sievorptive chromatography. Intervent dilution chromatography, as well as the recently defined ion filtration chromatography (2), are specific modes of the general process of sievorptive chromatography. Although ion filtration and intervent dilution chromatography are conceptually distinct, in practice both processes may occur within the same column, as happens when ion filtration chromatography is used to "desalt" an enzyme sample.The chromatographic system that is introduced has broad utility and may be adjusted for a selective or complete dissociation of biological complexes. To illustrate the power of the new chromatographic approach, conditions were found to separate ribosomal proteins from ribosomal RNA, one of the strongest macromolecular complexes encountered in biochemistry. As a historical framework for this stu...

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Ion filtration chromatography: A powerful new technique for enzyme purification applied to E. coli alkaline phosphatase

Cited by 46 publications

References 10 publications

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase

Multiple Forms of DNA‐Dependent RNA Polymerase I from Immature Chick Liver

Intervent Dilution Chromatography: Concept for Separation of Strongly Interacting Macromolecules

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