Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities.

Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. We show that the hnRNP C proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.     

Purification of commercial preparations of NADP+ from AMP contamination

A method for purification of commercial preparations of NADP+ from AMP contamination is described. The purification procedure includes one-step anion-exchange chromatography on Dowex-1 (formate) and results in a highly purified salt-free coenzyme with a yield of 70-80%. The chromatography conditions have been selected allowing for complete separation of AMP from NADP+ in a HCOOH concentration gradient. This is followed by NADP+ elution with 1.5 M HCOOH containing HCOOK at a concentration at which the salt remains in solution during subsequent precipitation and washing of NADP+ with acetone. An addition of HCOOK is necessary to reduce the coenzyme elution volume that is important for further precipitation of NADP+ with acetone.     

Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.     

hnRNP A1 and hnRNP F modulate the alternative splicing of exon 11 of the insulin receptor gene

Exon 11 of the insulin receptor gene (INSR) is alternatively spliced in a developmentally and tissue-specific manner. Linker scanning mutations in a 5' GA-rich enhancer in intron 10 identified AGGGA sequences that are important for enhancer function. Using RNA-affinity purification and mass spectrometry, we identified hnRNP F and hnRNP A1 binding to these AGGGA sites and also to similar motifs at the 3' end of the intron. The hnRNPs have opposite functional effects with hnRNP F promoting and hnRNP A1 inhibiting exon 11 inclusion, and deletion of the GA-rich elements eliminates both effects. We also observed specific binding of hnRNP A1 to the 5' splice site of intron 11. The SR protein SRSF1 (SF2/ASF) co-purified on the GA-rich enhancer and, interestingly, also competes with hnRNP A1 for binding to the splice site. A point mutation -3U→C decreases hnRNP A1 binding, increases SRSF1 binding and renders the exon constitutive. Lastly, our data point to a functional interaction between hnRNP F and SRSF1 as a mutant that eliminates SRSF1 binding to exon 11, or a SRSF1 knockdown, which prevents the stimulatory effect of hnRNP F over expression.     

Ni2+-Based Immobilized Metal Ion Affinity Chromatography of Lactose Operon Repressor Protein from Escherichia Coli

Abstract

A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni²⁺-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.     

Purification of murine oncornaviral phosphoproteins using alkyl agarose derivatives..

Methods for the purification of both murine mammary tumor (type B) and murine leukemia (type C) oncornaviral phosphoproteins are described, in which chromatography on alkyl-agarose derivatives is used as the primary fractionation step. Gel filtration or ion exchange chromatography on phosphocellulose was the only subsequent step required for the purification of the type B and type C viral proteins, respectively. The two-step protocols also resulted in the co-purification of a low molecular weight core protein from each virus. Recoveries of the viral proteins purified by this method, based on per cent contribution of individual polypeptides to total virion proteins, were 70% or greater. Radioimmunocompetition analysis of the purified murine mammary tumor virus major core protein as well as analysis of the RNA binding properties of purified low molecular weight type C virus proteins suggests that neither antigenic reactivity nor specific RNA binding characteristics are altered by the purification protocols. The availability of these procedures should aid studies on the possible function and immunochemical properties of the native murine oncornaviral phosphoproteins and may also be extended to the purification of other oncornaviral proteins.     

A three-step procedure for the isolation of peptides derived from adenine nucleotide binding sites of enzymes labeled with p-fluorosulfonyl [14C]benzoyl-5'-adenosine.

A simple three-step procedure is described for the purification of a labeled peptide from a tryptic digest of the β-subunit of the F₁-ATPase after the enzyme had been inactivated with p-fluorosulfonyl-[¹⁴C]benzoyl-5′-adenosine. The procedure involves: (1) anion-exchange chromatography of a tryptic digest of the labeled β-subunit on diethylaminoethyl-Sephadex; (2) treatment of the peptides in the radioactive peak from the first step with 0.1m NaOH under conditions in which the ester bond in the label is hydrolyzed; and (3) anion-exchange chromatography of the treated peptides under conditions identical to those of the first step after removal of the NaOH by gel filtration. Cleavage of the ester bond in the second step releases adenosine and specifically introduces an additional negative charge onto the labeled peptide. Thus, it is resolved from the peptides that contaminate it in the third step.     

Purification and characterization of beta-adrenergic receptor mRNA-binding proteins

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.     

An improved method for the purification of lignin peroxidases from Phanerochaete chrysosporium INA-12: properties of two major isoforms.

1. Phanerochaete chrysosporium INA-12 secretes several lignin peroxidase isoenzymes. This paper reports an improved procedure for the purification of the different isoforms compared to those previously described. 2. Lignin peroxidases are first concentrated and prefractionated on fast-flow ion-exchangers which avoid concentration by ultrafiltration and dialysis. 3. Further purification is achieved by hydrophobic interaction chromatography and anion-exchange FPLC. 4. Two major forms were purified to homogeneity. Kinetic measurements and protein characterization (isoelectric points, phosphate content) suggest that they are similar to those produced by P. chrysosporium BKM strain.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.     

Large-scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA-cellulose

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single-stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large-scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53-55 kDa) can be purified, which are structurally and antigenically related to the 'canonical' hnRNP core proteins (34-43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22-28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.     

Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells

Recently we have identified a mitogen-activated S6 kinase from Swiss 3T3 cells (Jen?, P., Ballou, L. M., Novak-Hofer, I., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 406-410). Here we describe the detailed purification of this enzyme from high-speed supernatants (400,000 x g) of vanadate-treated cell extracts. The enzyme is purified through six sequential steps including cation- and anion-exchange, sizing, and affinity chromatography. At each step, the enzyme behaves as one entity and, on the final step of purification, is revealed on silver-stained sodium dodecyl sulfate-polyacrylamide gels as a single protein of Mr 70,000. As reported earlier, the overall purification factor is 3,000-fold, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein. However, recovery of total activity is only 0.2%. This large loss of activity appears to be due to freeze-thawing the enzyme between each step of purification. The purified kinase does not phosphorylate casein, histones 2A and 3S, or phosvitin. It has a Km for ATP of 28 microM and a broad optimum for Mg2+ between 5 and 20 mM. Mn2+ does not affect the basal level of kinase activity, and at concentrations as low as 1 mM, it completely suppresses the effect of 20 mM Mg2+ on kinase activity. The relationship of this enzyme to two other purified S6 kinases is discussed.     

Purification of oligo dG-tailed Okayama-Berg linker DNA fragments by oligo dC-cellulose chromatography

A simple procedure for preparation of oligo dG-tailed DNA fragments is presented. The fragments are first purified by ultracentrifugation through sucrose gradients at low salt concentration. Appropriate gradient fractions are then adjusted to 1 M NaCl and immediately applied to a column of oligo dC-cellulose equilibrated in buffered 1 M NaCl at 4 degrees C. Fragments are eluted with water at room temperature. Passage through the column achieves, in one step, the concentration and purification of oligo dG-tailed DNA fragments free from sucrose.     

Purification of a -hydantoinase using a laboratory-scale Streamline phenyl column as the initial step

A -hydantoinase from Thermus sp. was overexpressed in Escherichia coli and purified to homogeneity for subsequent crystallization. The purification was performed with hydrophobic interaction chromatography as the capture step followed by anion-exchange chromatography and gel permeation chromatography as intermediate purification and polishing steps, respectively. The hydrophobic interaction step was done in fluidized bed mode in a laboratory-scale Streamline column made from conventional laboratory equipment. The whole purification protocol could be finished within one day. The purified enzyme crystallizes. The crystals are suitable for X-ray protein structure analysis and diffract to at least 2.3 Å resolution. Complete data sets have been measured up to 2.6 Å resolution. The X-ray structure is currently being solved.     

Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments

A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogeneous after purification and exhibits a molecular mass of approximately 85 kDa, as determined by SDS/PAGE. From its chromatographic behaviour on hydroxyapatite, and from Western-blotting experiments using isoenzyme-specific antibodies, we were able to identify the bovine rod outer segment protein kinase C as being of the alpha or type-III form. The purified protein kinase C has a specific activity of 1066 nmol 32P.min-1.mg protein-1, and shows a 30-fold activation upon the addition of the effectors Ca2+, PtdSer and 1,2-diacylglycerol. Arachidonic acid and linoleic acid were also found to enhance significantly the activity of the purified enzyme.     

Modification of pyruvate kinase activity by proteins from chicken liver

The partial purification of a protein fraction inhibiting pyruvate kinase isoenzymes is described. The fraction was isolated from the (NH4)2SO4 step of the purification procedure for pyruvate kinase isoenzymes from chicken liver (Eigenbrodt, E. & Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1033-1046) by extraction with 1N NaOH, acidification to pH 3, ethanol precipitation and chromatography of the supernatant on DEAE-cellulose. The inhibitor fraction was further purified by disc gel electrophoresis using a gel gradient from 10 to 25%; this procedure separated activating proteins from the inhibitor fraction. The inhibitor fraction inhibited the pyruvate kinase isoenzymes from chicken in the sequence of decreasing effect: M2 greater than L greater than M1. The inhibition was due to a decrease in the affinity for phosphoenolpyruvate. The inhibitor is stable against heating for 5 min in 1% sodium dodecyl sulfate at 100 degrees C; it is destroyed by pepsin digestion. The inhibitor fraction could be purified further only by dodecyl sulfate gel electrophoresis. This resulted in the separation of 2 inhibitors (Mr = 33,500 +/- 8500 and ca. 5000), an activator (Mr = 15,100 +/- 5200), and an unidentified protein (Mr = 27,000).     

Partial purification of androgen binding protein from bull epididymis

An androgen binding protein (ABP), with an equilibrium dissociation constant of 4.2 nM and a molecular weight of about 100 kDa, has been purified from bull epididymal extracts using a four-step procedure. These preliminary results underline the main difficulties encountered in the purification of this protein present at a very low concentration (i.e. 50-fold less than in rat or rabbit epididymides). Ammonium sulfate precipitation is not a suitable step due to the formation, in presence of salt, of insoluble material leading to a loss of ABP. Lipids, particularly phospholipids, might be implicated in this phenomenon. Several steps, including anion exchange in batch followed by concentration, affinity chromatography and HPLC gel filtration allowed us to obtain a 7667-fold purified protein with a 9% yield.     

Characteristics of a proteinase from ovarian fluid of the lumpsucker (Cyclopterus lumpus L.)

1. A proteinase has been isolated from the ovarian fluid of the lumpsucker (Cyclopterus lumpus). 2. The enzyme was purified essentially to homogeneity by a one step purification procedure using anion-exchange chromatography. 3. The mol. wt of the denatured enzyme is approximately 20,000 as judged by SDS-polyacrylamide gel electrophoresis. 4. The enzyme is inhibited by serine-proteinase inhibitors and acts in the manner of a trypsin-type proteinase both with respect to specific peptide substrates and enzyme inhibitors. 5. The lumpsucker proteinase exhibits low general proteolytic activity but acts effectively on the specific chromogenic peptide substrates.     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).