General Method For Purification Research Articles

Easy and efficient protocol for purification of recombinant peptides

Peptide synthesis and purification remains a challenge. Low abundance leads to small yields when peptides are purified from natural sources. On the other hand, synthetic methods are limited by the chemical properties of the amino acids and the concurrent aggregation of peptides. In this paper, we report a versatile, high yielding and general purification method for randomly chosen recombinant peptides of variable sizes (ranging from ∼1.7kDa to ∼10kDa). Expressed as fusion proteins with commonly used tag proteins, these peptides are cleaved by ‘PreScission protease’ in a volatile buffer that makes concentration and recovery of the peptide easy. Separation of the cleaved peptide is achieved by selective precipitation of the larger tag protein with acetonitrile; leaving the peptide in solution. Our protocol can be used to generate a wide variety of peptides in significant quantities for biochemical, biophysical and physiological studies.

Well-Defined Polyisoprene-b-Poly(acrylic acid)/Polystyrene-b-Polyisoprene-b-Poly(acrylic acid) Block Copolymers: Synthesis and Their Self-Assembled Hierarchical Structures in Aqueous Media.

The synthesis and characterization of well-defined polyacid based block copolymers containing polyisoprene (PI) are reported. The challenge of maintaining the integrity of the polydiene while producing polyacid from the tert-butyl ester precursor is addressed in this communication. A general purification method was also developed, taking advantage of the different polarities of each block. The polystyrene-b-polyisoprene-b-poly(acrylic acid) (PS-b-PI-b-PAA) triblock terpolymers form multicompartmental micelles via aqueous self-assembly. Our work reveals the morphological consequences of unique balances among global and local interactions.

Pharmaceutical study of Yashadabhasma

Background:Rasashastra is a branch which deals with the pharmaceutics of Rasaoushadhis. Bhasmas are one among such Rasaoushadhis which are known for their low doses and fast action. A verse from Rasaratnasamuchchaya says that the bhasma prepared by using Mercury as media is of best quality.Materials and Methods:Following this principle, Yashadabhasma (Zinc calx) was prepared by subjecting it to Samanya shodhana (general purification method for all metals), Vishesha shodhana (specific putification method for Zinc), Jarana (roasting) and Marana (incineration) with Parada(Mercury) as a media under Gajaputa (classical heating system with 1000 cowdung cakes).Results and Conclusion:Yellow colored Yashadabhasma which passed all the classical bhasmaparikshas (tests for properly prepared calx) was obtained after two putas. The bhasma did not pass Nishchandratva(free from shining particles) test after 1stputa but was passed after giving it 2ndputa.

NMR Solution Structure of the Mitochondrial F 1β Presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F 1β subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F 1β presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F 1β presequence formed an α-helical structure in membrane mimetic environments such as SDS and DPC micelles (∼50% α-helix), and in acidic phospholipid bicelles (∼60% α-helix). The NMR solution structure of the F 1β presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic α-helix (residues 4–15) and a C-terminal α-helix (residues 43–53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic α-helix forms the putative Tom20 receptor binding site, whereas the C-terminal α-helix is located upstream of the mitochondrial processing peptidase cleavage site.

An efficient purification process for sweet potato beta-amylase by affinity precipitation with alginate

β-amylases are used in production of maltose syrup. It is shown that sweet potato β-amylase can be purified by affinity precipitation with alginate with 80% activity yield and 44 fold purification. SDS-PAGE of the purified protein showed a single band and a subunit weight of 50 kDa. Preliminary data with soybean and barley enzymes indicate that this may be a general method for purification of β-amylases.

Histidine-Tagged Ubiquitin Substitutes for Wild-Type Ubiquitin in Saccharomyces cerevisiae and Facilitates Isolation and Identification of in Vivo Substrates of the Ubiquitin Pathway

A general method for purification of any substrate of the ubiquitin pathway, the major eukaryotic proteolytic pathway, should utilize the common characteristic of covalent linkage of ubiquitin to substrate lysyl residues. The utility of a N-terminal histidine-tagged ubiquitin (HisUb) for in vivo conjugation and isolation of ubiquitinated proteins by metal chelation chromatography is conditioned by the requirement that HisUb conjugate to the same set of proteins as wild-type ubiquitin. Stringent in vivo tests with Saccharomyces cerevisiae strains expressing ubiquitins only from plasmids were performed to show that HisUb could substitute for wild-type ubiquitin. The utility of HisUb as a method for purification of proteins ubiquitinated in vivo was demonstrated by metal chelation chromatography of yeast extracts expressing HisUb and immunoblotting for Rpb1, the largest subunit of RNA polymerase II. A fraction of Rpb1 was present in the ubiquitinated form in vivo. The ability to use HisUb expression in transgenic organisms that retain expression of their endogenous ubiquitin genes was demonstrated through transgenic Arabidopsis thaliana expressing HisUb or its variant HisUbK48R. UbK48R is a version of ubiquitin capable of conjugation to proteins, but cannot serve as an attachment site for ubiquitin via the major in vivo interubiquitin linkage. Whereas transgenic plants expressing HisUb showed insignificant enrichment of ubiquitinated proteins, transgenic Arabidopsis lines expressing HisUbK48R gave a much better yield.

Fractionation of low molecular weight polyethylene by high‐pressure soxhlet extraction

AbstractThe use of quantitative, high‐pressure Soxhlet extraction for the fractionation of low molecular weight polyethylene samples is described. Liquid carbon dioxide was found to be a suitable solvent for the lowest molecular weight hydrocarbons but failed to solubilize hydrocarbons with molecular weights greater than C‐40—C‐50. Liquid pentane was found to be an effective solvent for hydrocarbons that were insoluble in liquid CO2. Careful, stepwise adjustment of the extraction solvent temperature produced polymer fractions with molecular weight distributions substantially narrower than those of the parent materials. Polymer fractions with molecular weights up to C‐90 were analyzed by high‐temperature gas chromatography. These analyses demonstrated the effectiveness of the technique in fractionating polymers according to molecular weight. Further evidence was provided by thermal analysis of the fractions that indicated melting‐point transitions that were much sharper than those of the parent materials. High‐pressure Soxhlet extraction offers considerable potential as a general method for purification and fractionation of synthetic and natural polymers. © 1993 John Wiley & Sons, Inc.

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.

A general method for purification of H1 histones that are active for repression of basal RNA polymerase II transcription

H1 histones were purified from extracts of salt-treated nuclei as a co-product of RNA polymerase II transcription factors from both Drosophila embryos and HeLa cells by a simple and general method. This procedure was also used to purify H1 as co-product of the core histones from calf thymus. The key steps in this purification exploit the solubility of H1 in 2.26 m ammonium sulfate and the chromatographic properties of the highly charged H1 molecules on a phenyl-Sepharose resin. H1 that is prepared by this procedure is active for in vitro repression of basal RNA polymerase II transcription. This method provides a new means of purifying H1 by a mild procedure that is likely to be generally useful for studies of transcription and chromatin structure.

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.

Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent

We describe a general purification method for recombinant proteins based upon the selective interaction between a poly-histidine peptide, which is fused to the protein of interest, and a novel metal chelate adsorbent. The principle of the technique is illustrated with mouse dihydrofolate reductase. DNA elements coding for adjacent histidines were fused to the mouse dihydrofolate reductase gene. Subsequent expression in E. coli resulted in the production of hybrid proteins that could be purified by immobilized metal ion affinity chromatography, followed by removal of the histidine affinity peptide with carboxypeptidase A.

Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.

General method for the purification of lipids for surface pressure studies : Application to monogalatosyldiglyceride

In order to prepare lipids free of surface-active contaminants ad suitable for the measurement of surface pressure isotherms as general purification method has been developed involving preparative thin-laye chromatography. A detailed description of the procedure and blank experiments carried out with a monolayer trough are presented. The method provides a rapid purification of various classes of lipids, the whole procedure taking less than 1 day. It gives highly reproducible results, better than 2 Å 2 per molecule at any surface pressure. As an example of an application, the results obtained with a commercial sample of monogalactosyldiglyceride, a major plant lipid, are reported.

A general method for purification of deoxycytidine kinase

Deoxycytidine kinase from a variety of animal tissues binds to Cibacron Blue 3G-A coupled to Sepharose CL-6B. The enzyme can be selectively eluted with ATP to yield single-step purifications of 17-89-fold. The affinity adsorbent is re-usable.

Lectin purification using formalinised erythrocytes as a general affinity adsorbant.

MANY carbohydrate-binding proteins, called lectins, purified from plant extracts1, are proving useful for the study of protein-carbohydrate interactions, cell-surface-initiated differentiation of cells and the topography of cell surface constituents1,2. A general method for purification of lectins would markedly augment this work. Affinity chromatography has been successful for purification of some lectins2 although others require conventional chemical fractionation. Purification may be simplified by the availability of commercial carbohydrate polymer adsorbants: for example Sephadex G-503 for concanavalin A and Sepharose 4B for the carbohydrate-binding protein from Dictyostelium discoideum4.