Affinity-purification of Transferrin-binding protein B under nondenaturing conditions

The commonly used purification procedures for Transferrin-binding protein B (TbpB) are based on an affinity chromatography step using resins onto which human transferrin had been immobilized. These protocols involve protein elution using denaturing buffer solutions. Here we present an improved protocol which permits protein elution under nondenaturing conditions using chelating agents such as phosphate or compounds containing a pyrophosphate group. Furthermore, isothermal titration calorimetry experiments of the purified protein with holotransferrin have been shown to be a reliable method to assess the purity and activity of the purified material.     

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions

Salmon nasal cartilage was micronized in ethanol using a rotor–stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.     

Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions

The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.     

Recombinant NY-ESO-1 cancer antigen: production and purification under cGMP conditions

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.     

Polycistronic coexpression and nondenaturing purification of histone octamers

Histone octamers are the basic building blocks of chromatin and are platforms for diverse genetic mechanisms. We report a simple method for preparing recombinant histone octamers by overexpressing all four histones from a single polycistronic vector followed by standard chromatography under native conditions. This approach reduces the time needed for the octamer preparation to a single day and should be applicable to making a variety of unmodified and modified histone octamers.     

Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions

Proteomes are dynamic, i.e., the protein components of living cells change in response to various stimuli. Protein changes can involve shifts in the abundance of protein components, in the interactions of protein components, and in the activity of protein components. Two-dimensional gel electrophoresis (2-DE) coupled with peptide mass spectrometry is useful for the analysis of relative protein abundance, but the denaturing conditions of classical 2-DE do not allow analysis of protein interactions or protein function. We have developed a nondenaturing 2-DE method that allows analysis of protein interactions and protein functions, as demonstrated in our analysis of the cytosol and crude membrane fractions of the facultative anaerobe Shewanella oneidensis MR-1. Our experiments demonstrate that enzymatic activity is retained under the sample and protein separation methods described, as shown by positive malate dehydrogenase activity results. We have also found protein interactions within both the soluble and membrane fractions. The method described will be useful for the characterization of the functional proteomes of microbial systems.     

Rapid purification of recombinant human lipocortin-I secreted fromSaccharomyces cerevisiae

Human lipocortin-I was expressed as a secretory product bySaccharomyces cerevisiae harboring an expression system consisting ofGAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys²⁶.     

Preparation of highly 3H-labeled S-100 protein under nondenaturing conditions

A method for obtaining a tritium-labeled S-100 protein of high specific radio-activity (〉~ 10 Ci/mmol) under mild conditions is described. The method is based on the reductive methylation of lysine residues; the labeling procedurs does not appreciably alter the physical and chemical properties of 8–100 protein, as measured by studies of intrinsic fluorescence enhancement, ⁴⁵Ca binding, electrophoretic mobility, titrations of sulfydryl groups, and intramolecular crosslinking of S-100 via disulfide bond formation. Alternative labeling procedures based on chemical or enzymatie iodination with ¹²⁵I, invelving the use of powerful oxidizing agents, cause an irreversible exidation of the sulfydryl groups and affect the above-mentioned properties of the S-100 protein.     

Backbone dynamics of the monomeric lambda repressor denatured state ensemble under nondenaturing conditions

Oxidizing two native methionine residues predominantly populates the denatured state of monomeric lambda repressor (MetO-lambdaLS) under nondenaturing conditions. NMR was used to characterize the secondary structure and dynamics of MetO-lambdaLS in standard phosphate buffer. 13Calpha and 1Halpha chemical shift indices reveal a region of significant helicity between residues 9 and 29. This helical content is further supported by the observation of medium-range amide NOEs. The remaining residues do not exhibit significant helicity as determined by NMR. We determined 15N relaxation parameters for 64 of 85 residues at 600 and 800 MHz. There are two distinct regions of reduced flexibility, residues 8-32 in the N-terminal third and residues 50-83 in the C-terminal third. The middle third, residues 33-50, has greater flexibility. We have analyzed the amplitude of the backbone motions in terms of the physical properties of the amino acids and conclude that conformational restriction of the backbone MetO-lambdaLS is due to nascent helix formation in the region corresponding to native helix 1. The bulkiness of amino acid residues in the C-terminal third leads to the potential for hydrophobic interactions, which is suggested by chemical exchange detected by the difference in spectral density J(0) at the two static magnetic fields. The more flexible middle region is the result of a predominance of small side chains in this region.     

Extraction of proteins from the large subunit of bovine mitochondrial ribosomes under nondenaturing conditions

The 55 S mammalian mitochondrial ribosome (referred to hereafter as "mitoribosome") is protein-rich, containing nearly twice as much protein as the Escherichia coli ribosome. In order to produce soluble mitochondrial proteins and protein-deficient subribosomal particles for use in functional and structural studies, the proteins of bovine mitoribosomes were extracted by washing in a series of buffers containing increasing concentrations of LiCl as the only chaotropic agent. LiCl disruption is used in order to preserve the solubilized proteins in a substantially "native" configuration. The extraction mixtures were characterized by sucrose density gradient analysis and the compositions of the stripped protein and residual pellet fractions were determined by two-dimensional polyacrylamide gel electrophoresis. In order to analyze the behavior or individual proteins, the intensity of Coomassie blue stain for each protein was normalized against the intensity of stain for the same protein in a control sample. Buffers with 1, 2, and 4 M LiCl each extract a specific subset of mitoribosomal proteins, while another group of proteins remains in the residual pellet fraction. Although very few proteins are detected in only one condition, most proteins are specifically enriched in one fraction. This LiCl procedure, therefore, produces fractionated groups of mitoribosomal proteins which can be used directly as a source for those proteins in which they are enriched, or they can be used as a starting point in further purification procedures. In contrast to results with E. coli ribosomes, several mitoribosomal proteins remain core-associated, indicating a different structural organization in these ribosomes.     

Methionine oxidation of monomeric lambda repressor: the denatured state ensemble under nondenaturing conditions

Although poorly understood, the properties of the denatured state ensemble are critical to the thermodynamics and the kinetics of protein folding. The most relevant conformations to cellular protein folding are the ones populated under physiological conditions. To avoid the problem of low expression that is seen with unstable variants, we used methionine oxidation to destabilize monomeric lambda repressor and predominantly populate the denatured state under nondenaturing buffer conditions. The denatured ensemble populated under these conditions comprises conformations that are compact. Analytical ultracentrifugation sedimentation velocity experiments indicate a small increase in Stokes radius over that of the native state. A significant degree of alpha-helical structure in these conformations is detected by far-UV circular dichroism, and some tertiary interactions are suggested by near-UV circular dichroism. The characteristics of the denatured state populated by methionine oxidation in nondenaturing buffer are very different from those found in chemical denaturant.     

One-step fractionation method for isolating H1 histones from chromatin under nondenaturing conditions

By combining conventional methods of chromatin preparation with ion-exchange chromatography in CM-Sephadex C-25, we have been able to isolate and fractionate histone H1 variants from different sources. This method of fractionation is very simple and allows one to obtain, very rapidly, large amounts of these histones in a native nondenatured conformation.     

Typing dinucleotide repeats under nondenaturing conditions with single-base resolution and high sizing precision

Dinucleotide repeats are genetic markers that are useful for many purposes, including genetic epidemiology, population genetics, and genetic diagnostics. The accuracy of analyses based on dinucleotide repeat polymorphisms is highly dependent on the success achieved in minimizing genotyping errors. Genotyping errors in dinucleotide repeat typing may arise for various reasons, including polymerase chain reaction (PCR) processing errors and the use of unsuitable electrophoretic conditions for resolving amplification products (i.e., lack of single-base resolution and inadequate precision in allele sizing). We have recently described a nondenaturing electrophoretic system useful for detecting PCR processing errors that lead to misidentification of heterozygotes as homozygotes in (AC)_n repeat typing. Here, we show that this system also allows resolution of (AC)_n repeats in native conditions with single-base resolution and high sizing precision, on the basis of an analysis of seven human (AC)_n repeats ranging in size from 72 to 217 bp. This PAGE system is thus also useful for reducing the likelihood both of allele misidentification due to the absence of single-base resolution and of inaccuracies in allele sizing due to anomalous electrophoretic migrations among the alleles within an (AC)_n repeat.     

A new method for the purification of 30S ribosomal proteins from Escherichia coli using nondenaturing conditions.

A new method for the purification of Escherichia coli (A19) 30S ribosomal proteins has been developed that avoids the use of denaturing conditions such as urea, acetic acid, and lyophilization. In this way the majority of the proteins from the small ribosomal subunit can be obtained in 5--100 mg quantities and at greater than or equal to 90% purity. This has been achieved by the initial "splitting" of the proteins into two main groups with LiCl followed by fractionating on ion-exchange and gel-filtration columns, in the absence of urea and in the presence of salt. The proteins prepared by this nondenaturing procedure were soluble at high ionic strength and less soluble, being aggregated, at low salt concentrations. This behavior was exactly the opposite of that exhibited by proteins prepared with methods using denaturing conditions. These new methods have enabled additional ribosomal RNA-binding proteins to be found and potential protein-proteins complexes to be isolated. Preliminary evidence that these proteins may retain a more native structure is presented.     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

Expression and purification of recombinant active prostate-specific antigen from Escherichia coli

Human prostate-specific antigen (PSA), a 33 kDa serine protease with comprehensive homology to glandular kallikrein, is secreted from prostatic tissue into the seminal fluid and enters into the circulation. The level of PSA increases in the serum of patients with prostatic cancer and hence is widely employed as a marker of the disease status. In particular, an enzymatically active PSA that is a form cleaved at the N-terminal seven-amino-acids prosequence, APLILSR, of proPSA may play an important roll in the progression of prostate cancer. Thus, the presence of the active form would selectively discriminate the cancer from benign prostatic hyperplasia. In this study, we developed a convenient purification method for the acquisition of active PSA and proPSA. Recombinant proPSA and active PSA were expressed directly in Escherichia coli, easily and efficiently isolated from inclusion bodies, refolded, and purified. Moreover, the enzymatic activity of the recombinant active PSA was confirmed as serine protease using chromogenic chymotrypsin substrate. This purified active PSA could be further applied to scrutinize the biological or conformational characteristics of the protein and to develop specific diagnostic and/or therapeutic agents against prostate cancer.