Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Purification and properties of a host cell protein required for poliovirus replication in vitro

A host cell protein required for poliovirus RNA-dependent RNA replicase activity in vitro has been purified several thousand-fold from an uninfected HeLa cell postmitochondrial supernatant. A single protein of apparent Mr = approximately 67,000 daltons and pI 6.3 is associated with this "host factor" activity. Poly(U)-Sepharose chromatography of the template-dependent replicase isolated from poliovirus-infected cells results in the complete loss of replicase activity if a salt gradient is used to develop the column. Host factor elutes early in the salt gradient and restores replicase activity to protein fractions eluted later in the gradient. The host factor, estimated to be present at 50,000-100,000 copies/cell, interacts physically with replicase.     

Affinity chromatography of protein kinase C-phorbol ester receptor on polyacrylamide-immobilized phosphatidylserine

An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.     

Preparation and properties of a complex from rat liver of polyribosomes with components of the cytoskeleton.

Gel filtration with 1% agarose (Bio-Gel A-150m) separates polyribosomes bound to microsomal membranes from 'free' polyribosomes when these fractions are prepared by standard centrifugal techniques. However, when polyribosomes contained in an unfractionated postmitochondrial supernatant are run on an identical column, over 90% of the total polyribosomes are present as aggregates, designated 'membrane-cytomatrix', which are eluted in the column void volume. Polyribosomes are not released from these aggregates on removal of microsomal phospholipids by treatment of postmitochondrial supernatant with 1% Triton X-100, a neutral detergent. The aggregates are disrupted by the usual ultracentrifugation techniques used in subcellular fractionation. After treatment of membrane-cytomatrix with Triton X-100 to remove phospholipids and membrane proteins, 58% of the polyribosomes still remain associated with protein-containing complexes in the form of a cytomatrix and are not 'free'. Preparations of both membrane-cytomatrix and cytomatrix are capable of sustained protein synthesis. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the cytoskeletal proteins actin and myosin are present in the cytomatrix. Incubation of cytomatrix preparations with the actin-depolymerizing agent deoxyribonuclease I caused release of the polyribosomes. Polyribosome release by deoxyribonuclease I was prevented by prior incubation with phalloidin, which is known to stabilize F-actin. Thus polyribosomes are associated with cytoskeletal elements in rat liver, and this association is dependent on polymeric forms of actin.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

A simple procedure for the isolation and purification of protamine messenger ribonucleic acid from trout testis.

Preparation of milligram quantities of purified poly(A)+ (polyadenylated) protamine mRNA from trout testis tissue was accomplished by a simple procedure using gentle conditions. This involves chromatography of the total nucleic acids isolated by dissociation of polyribosomes with 25 mM-EDTA to release messenger ribonucleoprotein particles and deproteinization of the total postmitochondrial supernatant with 0.5% sodium dodecyl sulphate in 0.25 M-NaCl by binding it to a DEAE-cellulose column. Total RNA was bound under these conditions, and low-molecular-weight RNA, lacking 18S and 28S RNA, could be eluted with 0.5 M-NaCl and chromatographed on oligo(dT)-cellulose columns to select for poly(A)+ RNA. Further purification of both the unbound poly(A)- RNA and the bound poly(A)+ mRNA on sucrose density gradients showed that both 18S and 28S rRNA were absent, being removed during the DEAE-cellulose chromatography step. Poly(A)- RNA sedimented in the 4S region whereas the bound poly(A)+ RNA fraction showed a main peak at 6S [poly(A+) protamine mRNA] and a shoulder in the 3-4S region. Analysis of the main peak and the shoulder on a second gradient showed that most of the main peak sedimented at 6S, whereas the shoulder sedimented slower than 4S. The identity of the poly(A)+ protamine mRNA was established by the following criteria: (1) purified protamine mRNA migrated as a set of four bands on urea/polyacrylamide-gel electrophoresis; (2) analysis of the polypeptides synthesized in the wheat-germ extract by starch-gel electrophoresis showed a single band of radioactivity which co-migrated exactly with the carrier trout testis protamine standard; and (3) chromatography of the polypeptide products on CM-cellulose (CM-52) showed the presence of three or four radioactively labelled protamine components that were co-eluted with the unlabelled trout testis protamine components added as carrier. The availability of large quantities of purified protamine mRNA should now permit a more thorough analysis of its physical and chemical properties.     

Purification of a delayed hypersensitivity-inducing protein from Listeria monocytogenes

Abstract Cell-envelope fragments were prepared from Listeria monocytogenes L242, serotype 4b. Delayed hypersensitivity (DH)-inducing proteins were extracted with deoxycholate and separated into two fractions by filtration through a Sephacryl S-200 column equilibrated with deoxycholate buffer. The second peak eluting from the Sephacryl column was fractionated using ion exchange chromatography on a DEAE Sepharose CL-6B column in the presence of 6 M urea. A purified 20 400-Da protein which induced DH against L. monocytogenes was obtained by isocratic elution. Three other DH-inducing fractions containing several protein bands were eluted by a gradient of potassium thiocyanate (KSCN) in urea buffer. Our results indicate that denaturing conditions can be employed for the fractionation and purification of DH inducing proteins from L. monocytogenes . In addition, it is suggested that the procedure described might also be useful for the purification of other antigens involved in cellular immune reactions.     

Purification of serine acetyltransferase,a component of a multienzyme complex,by immunoadsorption and selective dissociation of the complex

An immunoadsorbent column chromatography procedure utilizing antibody to one component of a multienzyme complex has been utilized for purification of the second component of this complex. Rabbit immunoglobulin G (IgG) specific for the O-acetylserine sulfhydrylase component of the multienzyme complex cysteine synthetase was linked to Sepharose 4B resin. A crude preparation of cysteine synthetase was bound to a column of IgG-Sepharose, other proteins were removed by washing, and the serine acetyltransferase component of the complex was eluted with 50 mmO-acetylserine, which dissociates the complex of the two enzymes. This purification step produces about a 400-fold increase in specific activity.     

Novel method for purification of staphylococcal enterotoxin A

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Use of Sepharose 4B for Preparative Scale Fractionation of Eukaryotic Messenger RNA's

A new technique using Sepharose 4B column chromatography for the partial purification of the total messenger RNA population of several animal tissues is described. The column when eluted with 0.1M sodium acetate, pH 5.0, containing. 001M EDTA, resolves a total nucleic acid extract into four major peaks. DNA is eluted at the void volume, followed successively by peaks of 18S ribosomal RNA, 4S transfer RNA and 28S ribosomal RNA. Ribonucleic acid containing mRNA activities is eluted after the DNA peak but immediately before the 18S rRNA peak. Hence the column enables quantitative removal of DNA, 5S RNA, tRNA and 28S rRNA from the majority of total cellular mRNA's. Partial segregation of mRNA's in the column is also demonstrated. The method does not require the isolation of polysomes as the initial procedure in mRNA isolation and is readily adaptable to large scale preparations. One hundred mg of total nucleic acid extracted from whole tissues can be fractionated on a 5 × 100 cm Sepharose 4B column. Recovery of total mRNA activities ranges between 60–70% and purification with respect to the total cell extract is 7 to 8 fold.     

Novel method for purification of staphylococcal enterotoxin A.

A novel single-step procedure for the purification of staphylococcal enterotoxin A (SEA), namely, dye ligand affinity chromatography with the triazine dye Red A, was developed. SEA purified by this method produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield from 5 liters of culture supernatant was 0.113 g, corresponding to an overall yield of 55%. In some instances, purification of SEA from culture supernatants by dye ligand affinity chromatography produced two enterotoxin peaks that could be eluted from the column with 300 and 500 mM phosphate buffer (pH 6.8). Enterotoxin from these peaks produced a single band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but multiple bands were observed on isoelectric focusing gels. This method of purification represents a significant improvement in time, yields, and purity of enterotoxin over previously published purification methods.     

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.     

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.     

Improved Method for Prufication of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase from Bovine Cerebral White Matter

Abstract: An improved procedure of the solubilization and purification of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M-ammonium acetate containing 10 mM-Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X-100 containing 10 mM-Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X-100 and 1 M-ammonium acetate mixture containing 10 mM-Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X-100-2 M-ammonium acetate and 4% Triton X-100-4 M-ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X-100-ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl-Sepharose CL-4B column chromatography was performed by eluting with a double-linear gradient of ammonium acetate and Triton X-100. In the second step, the fraction containing CNPase after Phenyl-Sepharose CL-4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X-100- I M-ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM-Sepharose CL-6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′-AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′-CAMP calculated from a Lineweaver-Burk plot was 3.13 mM.     

Isoelectric precipitation and gel chromatography for purification of monoclonal IgM

A preparative scale purification procedure of monoclonal IgM from hybridoma culture supernatant with high protein content is described. The procedure consists of three steps starting with ultrafiltration followed by isoelectric precipitation and gel chromatography. Cells and debris from culture supernatant were removed by microfiltration. The clarified supernatant was concentrated 400-fold in a hollow fibre ultrafiltration apparatus (cut off 100 000 daltons). The concentrate was titrated with dilute histidine/HCl buffer close to the isoelectric point of the IgM. Precipitated proteins were harvested by centrifugation, washed and redissolved. The protein fraction containing the IgM was further purified by gel chromatography on a Sephacryl S300 column. This procedure leads to product recovery of 40% and purity of 99% related to total protein.     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINO ACIDS: γ-AMINOBUTYRIC ACID RECEPTOR OF THE SHRIMP MUSCLE

Abstract— The total lipid extract of shrimp muscle ( Artemisia longinaris ) was precipitated with ether. The supernatant containing 95 per cent of the phospholipids and 50 per cent of the protein showed binding for L-[¹⁴C]glutamatc in the first peak of protein. The sediment, redissolved in chloroform-methanol was chromatographed on a Sephadex LH-20 column. A single peak was eluted in the chloroform (20-40 ml) having no lipid phosphorous and high affinity binding for [¹⁴C]GABA. The saturation was achieved at 1 mole per 80.000 g protein and the curve revealed a single type of binding site. The purification achieved was of about 4000-fold. There was no binding of L-[¹⁴C]glulamate to the ether precipitate. The specificity of the binding of [¹⁴C]GABA was further supported by competition experiments with bicuculline. picro-toxin and muscimol. It is suggested that the hydrophobic protein isolated by us represents the GABA receptor. The findings presented in the two papers of the series suggest that the excitatory and inhibitory receptor from crustacean muscle can be separated as two different proteins.     

The determination of folylpolyglutamate synthetase.

A relatively rapid radiochemical procedure for the determination of folylpolyglutamate synthetase activity is presented in this communication. The procedure is based on measurement of the incorporation of radioactive l-glutamate into tetrahydropteroylpolyglutamate on incubation with a tetrahydrofolate. After deproteinating the incubation mixtures with trichloroacetic acid, folate is separated from radioactive glutamate by an adaptation of a procedure generally employed in the isolation of folate from natural materials, i.e., adsorption on columns of charcoal from which it is subsequently eluted with aqueous-alcoholic ammonia containing mercaptoethanol and counted. The procedure is applicable to monitoring purification of the enzymes and to the study of their properties.The technique for separating a radioactive product of enzyme action from a radioactive precursor with a column of charcoal, that has been developed for this procedure is applicable also to other radiochemical enzyme determinations requiring the separation of an aromatic from an aliphatic metabolite.     

Use of ethanol-eluted hydrophobic interaction chromatography in the purification of serum amyloid A.

A two-step procedure for the purification of the acute-phase reactant serum amyloid A from serum is described. A hydrophobic interaction chromatography medium, octyl-Sepharose CL4B, eluted with increasing concentrations of EtOH was used as the first step in the purification. The concentrate from this step was applied to a gel filtration column of Sephacryl S-200 and eluted with 10% formic acid. The overall recovery of purified serum amyloid A from serum was 56%. This represents the first time that serum amyloid A has been purified without the use of high concentrations of guanidine or urea. The method presented could easily be scaled up to allow the purification of large quantities of serum amyloid A or readily adapted to the purification of other serum apolipoproteins.     

A rapid and simple method for the purification of rat liver RNase inhibitor

A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH₄)₂SO₄ and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity.