Preparative Electrofocusing for Industrial Applications

Abstract

This paper presents a case study of using a multicompartment isoelectric focusing apparatus to determine the isoelectric points and focus preparative quantities of brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3). A separation of PEGylated from unPEGylated forms of granulocyte colony stimulating factor (G-CSF) is described as well. Both BDNF and NT3 have substantially higher pI values in this system than is predicted from sequenced based modeling. Although PEGylated forms of G-CSF can be separated from the unPEGylated forms, separation of protein with differing degrees of PEGylation was not achieved. The paper additionally demonstrates that this technique can be used simultaneously as an analytical and preparative tool, eliminating the need for analytical IEF gels.     

ISOLATION OF AN ACID-SOLUBLE PROTEIN WITH LOW MOLECULAR WEIGHT FROM MONKEY BRAIN

Abstract— The isolation of a perchloric acid-soluble low molecular weight protein from brain of Macaca irus is reported. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel isoelectric focusing indicate that the protein is free of impurities. The molecular weight, as determined by gel filtration and sodium dodecyl sulphate gel electrophoresis, is shown to be 10,400 and 9900, respectively. This is in agreement with the value of 10,700 obtained from amino acid analysis. The protein contains 27 per cent acid amino acids and 15 per cent basic amino acids. However, the relatively high amide content gives the protein a neutral nature as shown by isoelectric point determination using gel isoelectric focusing.     

ISOLATION OF AN ACID-SOLUBLE BASIC PROTEIN FROM MONKEY BRAIN

—A basic protein, soluble in 0·1 m -perchloric acid, has been purified from brain of Macaca irus. The protein is homogeneous as indicated by ultracentrifugation, gel filtration, gel isoelectric focusing and gel electrophoresis at pH 2·9, 4·3 and 7·5. The molecular weight is estimated to be 16,000 by electrophoresis in sodium dodecyl sulphate–polyacrylamide gels. This result is in agreement with the value of 16,728 obtained from the amino acid analysis. The protein dimerizes under alkaline conditions. The predominant amino acid is glycine (15%) and the protein also contains 4% cysteine. The ratio of acidic to basic amino acids is 1·6, but a high amide content gives the protein a basic character. An isoelectric point of 9·5 is observed in gel isoelectric focusing.     

Isoelectric focusing in continuous-flow electrophoresis. I. Separation of mixed red blood cells of different species.

A noncommercial continuous-flow isoelectric focusing (CIF) apparatus which was formerly applied to separate mixtures of proteins was used to study the separation of red blood cells (RBC's) of different species. A mixture of human, mouse and rabbit erythrocytes, a good model for demonstration of cell separation by CIF, was completely separated into the three components. The separation was performed by isoelectric focusing in pH gradients, 3–10 and 5–7, using Ampholine carrier ampholytes at a field strength of 110 V/cm and a flow-through time of RBC's of 7 min. The isoionic points of human RBC's both determined by CIF and calculated from electrophoretic mobility measurements by extrapolation to zero electrophoretic mobility and zero ionic strength were found at pH 5.6–5.7. The method of CIF which is presently used to isolate a pure lysosomal fraction seems to be a valuable method for the separation of mixtures of cells or cell organelles.     

Separation of Component-Polypeptides of Glutenin by Isoelectric Focusing

1. By isoelectric focusing S-cyanoethyl glutenin was observed to be composed of various component-polypeptides having a pI spectrum in a pH range from 6 to 9.

2. During isoelectric focusing a precipitation zone was built up in the column in spite of the presence of 6 m urea. The amount of the precipitate formed was less with S-cyanoethyl glutenin than with S-sulfo glutenin.

3. S-Cyanoethyl glutenin was divided into eight fractions by isoelectric focusing. By starch-gel electrophoresis it was suggested that Fractions I, III and P were mainly composed of a single component.

4. Major N-terminal amino acids of Fractions I, III and P were phenylalanine, glycine and alanine, respectively. In the amino acid composition, distinct differences were observed in the respective fractions, especially in Fraction P. Fraction P showed a much higher content of basic amino acids and a lower content of glutamic acid in comparison with the other two.     

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Compositional relationships among electrophoretic isolates from cottonseed protein bodies

Minor differences exist among polypeptides isolated from cottonseed storage globulins with respect to nutritionally-limiting amino acids, although the composition of most isolates closely resembles that of the entire storage globulin fraction. Glutamate, arginine and aspartate are the most abundant amino acids; cysteine and methionine are the least abundant. In contrast to the 10 polypeptide bands in the SDS-PAGE pattern, isoelectric focusing produced a pattern of 49 components within a range of pH 4.5–9.5. A two-dimensional electrophoretic separation indicated that most of the polypeptides resolved by SDS-PAGE are heterogeneous with respect to charge. The high ratio of isoelectric to molecular weight forms may prove advantageous for efforts to genetically improve the quality of cottonseed protein.     

Purification and partial characterization of a cellular retinol-binding protein from human liver

A protein with binding specificity for retinol was purified from human liver. [³H]Retinol was added to liver extracts and the [³H]retinol-binding protein isolated by conventional chromatographic techniques including ion-exchange chromatography on DEAE-Sepharose, gel filtration on Sephadex G-75 and G-50 and preparative isoelectric focusing. The yield was 10–15% in different preparations and the degree of purification was about 3000-fold. The purified protein had a molecular weight of about 15 000 as estimated from both gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulphate and was homogeneous in several electrophoretic systems. Isoelectric focusing of the purified protein gave a doublet band. Only one fluorescent band at pH 4.70 was seen if the protein solution was incubated with excess retinol prior to isoelectric focusing. The isolated protein did not react with antiserum to the retinol-binding protein of plasma. The amino acid composition and the amino terminal amino acid sequence for the first sixteen amino acids of the purified protein differed significantly from that of the plasma retinol-binding protein.     

Solution isoelectric focusing for peptide analysis: comparative investigation of an insoluble nuclear protein fraction

In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, interfaced with mass spectrometry. An insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The two-dimensional separation method was combined with (18)O labeling for comparative analysis of protein expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the advantage that it can be accomplished after the (18)O labels are introduced. The labeled peptides can be mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line.     

Isolation and characterization of a hepatic metallothionein from mice.

Mice were given cadmium chloride orally for 18 weeks, after which they were given two subcutaneous injections of cadmium chloride. Cadmium-and zinc-containing proteins were obtained from livers by ultracentrifugation, Sephadex chromatography and isoelectric focusing. Metallothionein with apI of 4.2 at 8 degrees C was separated from other proteins (possibly including other forms of metallothionein) by the isoelectric procedure. The metallothionein with pI = 4.2 had a high E250, reflecting abundant Cd-SH bonds, in accord with amino acid analysis, which gave a cysteine content of 34.6 residues % and showed an absence of aromatic amino acids. These results indicated a very high purity of the form of metallothionein isolated after isoelectric focusing.     

Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja).

A novel variant of soybean Kunitz trypsin inhibitor (SKTI) was detected in 530 lines of wild soybean (Glycine soja). This variant showed an intermediate electrophoretic mobility between the Tia and Tic types. In isoelectric focusing polyacrylamide gel electrophoresis gels containing urea, this variant had a similar isoelectric point as that of Tia. The genetic analysis of SKTI bands in F2 seeds from crosses of the new variant type with Tia or Tic type showed that this variant type is controlled by a codominant allele at the SKTI locus. We propose the genetic symbol Tif for this novel variant. When the nucleotide sequence of the Tif gene was compared with those of other types of SKTI genes (Tia, Tib, and Tic), the sequence of Tif was identical to that of Tib with the exception of one A-->G transitional mutation occurring at position 676 of Tif. This mutation resulted in an amino acid change from Lys to Glu at the 178 residue. These results suggest that this variant is derived from Tib through a point mutation. In addition, we settled an inconsistency in the number of amino acid differences between Tia and Tib (eight or nine). Analysis of nucleotide and amino acid sequences revealed that Tib was different from Tia by nine amino acids.     

Electrophoretic isolation and partial characterization of a major secretory glycoprotein from the submandibular glands of the mouse

After either cholinergic or adrenergic stimulation of the submandibular glands of the mouse, a major protein of the incubation medium could be isolated by electrophoresis, designated the AM2 protein. About 5 per cent of the secreted proteins and 2.4 per cent of the secreted protein-bound sialic acid was recovered as the purified AM2 protein. The AM2 protein appeared to be electrophoretically pure in 7.5% polyacrylamide gel both at pH 8.9 and at pH 4.3. In sodium dodecyl sulfate-electrophoresis the molecular weight was estimated to be about 80 000 for the major component and about 40 000 for the minor component. By isoelectric focusing the isoelectric point has been determined to be 4.7. The amino acid analysis indicated Glx, Asx, Leu and Ala as the major amino acids, comprising 15.0, 10.6, 9.2 and 9.1 per cent of the amino acid residues, respectively. The ratio of the acidic amino acids and their amides (Glx plus Asx) to the basic amino acids (Lys plus Arg) was 2.2. The sugar analysis showed that the AM2 glycoprotein consists of 17.3 per cent of carbohydrate, with as major carbohydrate component glucosamine. The molar ratio of the sugars was Man : Gal : Glc : GlcNH2 : sialic acid = 2.3 : 1.0 : 4.7 : 9.8 : 2.9. Galactosamine could be detected as a trace component and fucose was not detectable.     

Peptide mapping of bovine and chicken cytochrome c by capillary isoelectric focusing with universal concentration gradient imaging

Capillary isoelectric focusing with universal concentration gradient imaging detection was used to separate and detect tryptic peptides from bovine and chicken cytochrome c. For a desalted sample of peptide angiotensin 2, the isoelectric point (pI) measured by the instrument agreed well with the pI calculated from amino acid pK values. For the cytochrome digests, correlations between measured and calculated pI values were imprecise because peak positions shifted slightly from test to test. This problem is thought to be caused by the inefficient desalting process used on the samples, leaving salt residues which caused distortion in the pH gradient during the focusing process. However, this system differentiated between the two cytochrome c's. The concentration gradient imaging detected peptides which contain no tyrosine and no tryptophan amino acids, which a UV absorption detector operating at 280 nm could not. The separation and detection steps took only 5–7 min because no mobilization was necessary after the focusing process.     

Polyacrylamide gel electrophoresis: recovery of non-stained and stained proteins from gel slices

Following electrophoresis or isoelectric focusing in gels of polyacrylamide the protein band of interest is cut out and placed above a sucrose gradient column, containing carrier ampholytes (Pharmalyte). By electrophoresis, isoelectric focusing or displacement electrophoresis the proteins migrate out of the gel slice and into the isoelectric focusing column for concentration and further purification. From this column, the proteins can be withdrawn and their isoelectric points determined. Even after staining with Coomassie Brilliant Blue at least some proteins can be recovered by this technique and used for further analyses, for instance amino acid determinations. The focusing in a pH gradient by carrier ampholytes can be replaced by an electrophoresis in a conductivity gradient column. However, in comparison with isoelectric focusing, this concentration technique has the drawback of not permitting further purification of the eluted protein.     

Analytical techniques for cell fractions. XXI. Two-dimensional analysis of serum and tissue proteins: multiple isoelectric focusing

Two-dimensional electrophoresis, using isoelectric focusing in one dimension and electrophoresis in sodium dodecyl sulfate in the second, yields the highest resolution separation of proteins and protein subunits thus far obtained. For general research use, for genetic screening, and for clinical studies, it is important to be able to make these analyses easily, relatively rapidly, and reproducibly. This paper describes a device and methods for casting up to 20 isoelectric focusing gels in parallel, for running them in the same apparatus, and for recovering them in preparation for the second-dimension separations.     

Human plasma angiotensinogen: A review of purification procedures

Summary The current status of the purification and characterization of human angiotensinogen is reviewed. One problem encountered in the past has been the copurification of a protein with similar properties. This protein has tentatively been designated alanine-protein. An efficient separation of angiotensinogen and alanine-protein was obtained on a zinc chelate column. Alanine-protein has been purified and its amino acid and carbohydrate composition determined. The COON-terminal amino acid and the NH₂-terminal amino acid were determined to be serine and alanine, respectively. Alanine-protein exhibited multiple forms on isoelectric focusing.This work was supported in part by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Wisconsin Heart Association.     

The preparation and properties of gonococcal pili.

Pili have been isolated from Neisseria gonorrhoeae by controlled homogenization followed by selective disaggregation in sucrose and purification by CsCl density gradient centrifugation. Pili from six gonococcal strains had buoyant densities of 1-30 to 1-31 g ml-1 on CsCl. The pili were immunologically distinct when tested with rabbit antisera to purified pili. The amino acid composition of pilin from strains P9 and 201 was very similar, consisting of 208 and 212 amino acid residues respectively giving molecular weights of 22 600 and 22352. The pili contained a high proportion (46%) of non-polar amino acids. Further analysis of strain P9 pili revealed the presence of 1 to 2 phosphate groups and 1 to 2 hexose groups per pilin subunit; no amino sugars were detected. Pili from strain P9 were resolved into two bands by equilibrium density gradient centrifugation or column isoelectric focusing, suggesting the presence of more than one kind of pilus.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.     

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.