Biomedical subjects

Tsutomu Arakawa

Publications and source records attributed to Tsutomu Arakawa.

At least 19 recordsLinked to original sources

PubMed2007

Effects of acid exposure on the conformation, stability, and aggregation of monoclonal antibodies.

Exposure of antibodies to low pH is often unavoidable for purification and viral clearance. The conformation and stability of two humanized monoclonal antibodies (hIgG4-A and -B) directed against different antigens and a mouse monoclonal antibody (mIgG1) in 0.1M citrate at acidic pH were studied using circular dichroism (CD), differential scanning calorimetry (DSC), and sedimentation velocity. Near- and far-UV CD spectra showed that exposure of these antibodies to pH 2.7-3.9 induced only limited conformational changes, although the changes were greater at the lower pH. However, the acid conformation is far from unfolded or so-called molten globule structure. Incubation of hIgG4-A at pH 2.7 and 3.5 at 4 degrees C over the course of 24 h caused little change in the near-UV CD spectra, indicating that the acid conformation is stable. Sedimentation velocity showed that the hIgG4-A is largely monomeric at pH 2.7 and 3.5 as well as at pH 6.0. No time-dependent changes in sedimentation profile occurred upon incubation at these low pHs, consistent with the conformational stability observed by CD. The sedimentation coefficient of the monomer at pH 2.7 or 3.5 again suggested that no gross conformational changes occur at these pHs. DSC analysis of the antibodies showed thermal unfolding at pH 2.7-3.9 as well as at pH 6.0, but with decreased melting temperatures at the lower pH. These results are consistent with the view that the antibodies undergo limited conformational change, and that incubation at 4 degrees C at low pH results in no time-dependent conformational changes. Titration of hIgG4-A from pH 3.5 to 6.0 resulted in recovery of native monomeric proteins whose CD and DSC profiles resembled those of the original sample. However, titration from pH 2.7 resulted in lower recovery of monomeric antibody, indicating that the greater conformational changes observed at this pH cannot be fully reversed to the native structure by a simple pH titration.

Acids↗

PubMed2007

Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart.

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.

Chromatography, High Pressure Liquid↗

PubMed2007

Effects of salts on protein-surface interactions: applications for column chromatography.

Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein-protein or protein-surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein-protein or protein-surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers.

Chromatography, Affinity↗

PubMed2006

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Ammonium Sulfate↗

PubMed2006

Improved performance of column chromatography by arginine: dye-affinity chromatography.

Arginine has been effectively used in various column chromatographies for improving recovery and resolution, and suppressing aggregation. Here, we have tested the effectiveness of arginine as an eluent in dye-affinity column chromatography using Blue-Sepharose, which binds enzymes requiring adenyl-containing cofactors (e.g., NAD). A common eluent, NaCl, showed a broad elution peak with low recovery of lactate dehydrogenase, at most approximately 60% using 2M salt. The recovery decreased as the NaCl concentration was either decreased or increased; i.e., the recovery was maximum at 2M. On the contrary, addition of arginine to the eluent resulted in more than 80% recovery above 0.5M and the recovery was nearly independent of the arginine concentration. The elution peak was much sharper with arginine, leading to elution of more concentrated protein solution. Successful elution of proteins bound to the ATP-agarose resins by arginine was also described.

Arginine↗

PubMed2006

Small molecule pharmacological chaperones: From thermodynamic stabilization to pharmaceutical drugs.

A great deal of attention has been paid to so-called amyloid diseases, in which the proteins responsible for the cell death and resultant diseases undergo conformational changes and aggregate in vivo, although whether aggregate formation is the cause or the result of the cell death is controversial. Recently, an increasing attention is given to protein folding diseases tightly associated with mutations. These mutations result in temperature-dependent misfolding and hence inactivation of the proteins, leading to loss of function, at physiological temperature; at low so-called permissive temperatures, the mutant proteins correctly fold and acquire functional structure. Alternatively, activation can be induced by use of osmolytes, which restores the folding of the mutant proteins and hence are called chemical chaperones. The osmolytes are compatible with macromolecular function and do stabilize the native protein structure. However, chemical chaperones require high concentrations for effective folding of mutant proteins and hence are too toxic in in-vivo applications. This limitation can be overcome by pharmacological chaperones, whose functions are similar to the chemical chaperones, but occur at much lower concentrations, i.e., physiologically acceptable concentrations. Although the research and clinical importance of pharmacological chaperones has been emphasized, the initial and central concept of osmolytes is largely ignored. Here we attempt to bridge the concept of osmolytes to applications of pharmacological chaperones.

Pharmaceutical Preparations↗

PubMed2006

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis.

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Amino Acid Sequence↗

PubMed2006

The secondary structure analysis of a potent Ser14Gly analog of antiAlzheimer peptide, Humanin, by circular dichroism.

The structure of a highly potent Ser14Gly analog of antiAlzheimer peptide, Humanin, was examined by circular dichroism (CD). The secondary structure is more disordered in water than in phosphate-buffered saline (PBS). The peptide structure in water is little dependent on both peptide concentration and temperature. On the contrary, the peptide structure was significantly different in PBS from the structure in water, which is more apparent at a higher peptide concentration and temperature. The observed different structure in PBS appears to be due to self-association of the peptide, which is enhanced by elevated temperature and, hence, via hydrophobic interactions. The wild-type Humanin also behaved similarly, i.e., it assumed a disordered structure in water but underwent conformational changes in PBS. Although high peptide concentrations for CD measurements are not encountered in vivo, the results suggest the tendency of the peptide to interact hydrophobically with other structures as well as with itself.

Alzheimer Disease↗

PubMed2006

Contribution of halophilic nucleoside diphosphate kinase sequence to the heat stability of chimeric molecule.

A halophilic nucleoside diphosphate kinase from a moderate halophile, Halomonas sp. 593 (593NDK), was found to be resistant to heat treatment, as indicated by the high level of activity recovery after heating at high temperatures. This is due to reversibility of thermal unfolding, not the high melting temperature, of the protein. The highly homologous NDK from non-halophilic organism, Pseudomonas aeruginosa, showed instability against heat treatment. Chimeric molecules consisting of each half of these two NDKs were constructed and characterized for their heat stability. The results showed that the N-terminal half of 593NDK contributes to the heat stability of the proteins. We discuss the possible reason for the observed difference in resistance to heat treatment between the 593NDK and PaNDK and between two chimeric proteins.

Amino Acids↗

PubMed2006

Aggregation suppression of proteins by arginine during thermal unfolding.

Arginine has been used to suppress aggregation of proteins during refolding and purification. We have further studied in this paper the aggregation-suppressive effects of arginine on two commercially important proteins, i.e., interleukine-6 (IL-6) and a monoclonal antibody (mAb). These proteins show extensive aggregation in aqueous buffers when subjected to thermal unfolding. Arginine suppresses aggregation concentration-dependently during thermal unfolding. However, this effect was not specific to arginine, as guanidine hydrochloride (GdnHCl) at identical concentrations also was effective. While equally effective in aggregation suppression during thermal unfolding, arginine and GdnHCl differed in their effects on the structure of the native proteins. Arginine showed no apparent adverse effects on the native protein, while GdnHCl induced conformational changes at room temperature, i.e., below the melting temperature. These additives affected the melting temperature of IL-6 as well; arginine increased it concentration-dependently, while GdnHCl increased it at low concentration but decreased at higher concentration. These results clearly demonstrate that arginine suppresses aggregation via different mechanism from that conferred by GdnHCl.

Antibodies, Monoclonal↗

PubMed2006

Expression of human tyrosine kinase, Lck, in yeast Saccharomyces cerevisiae: growth suppression and strategy for inhibitor screening.

We report the successful expression and detection of a phosphorylated form of human T cell tyrosine kinase, Lck, in Saccharomyes cerevisiae, which leads to growth suppression of the yeast cells. Expression of an inactive Lck mutant resulted in no phosphorylation and no growth suppression, indicating that cell growth inhibition by Lck is due to the activity of the kinase, consistent with the observed tyrosine-phosphorylation of the Lck and yeast host cell proteins. The addition of a known inhibitor of Lck to the cell culture resulted in recovery of cell growth expressing the active Lck, suggesting that the growth inhibition by lck gene expression can be used to screen inhibitors for the gene product. We have extended such approach to Tob, another potential therapeutic target.

Enzyme Inhibitors↗

PubMed2005

Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560.

Beta-lactamase from a moderately halophilic organism is expected to show salt-dependent stability. Here we examined the temperature-dependence of stability at different salt concentrations using circular dichroism (CD) and enzyme activity. NaCl showed opposing effects on melting temperature and reversibility of the thermal melting. Increasing NaCl concentration greatly increased the melting temperature from, e.g., 41 degrees C in the absence of NaCl to 61 degrees C in 3 M NaCl. Conversely, reversibility decreased from 92% to 0% in the corresponding NaCl solutions. When beta-lactamase was heated at different temperatures and NaCl concentrations, the activity recovery followed the reversibility, not the melting temperature. Heating beta-lactamase at 63 degrees C, slightly above the onset temperature of melting in 2 M NaCl and far above the melting in 0.2 M NaCl, showed a much greater recovery of activity in 0.2 M NaCl than in 2 M NaCl, again consistent with the reversibility of melting.

Circular Dichroism↗

PubMed2005

Effective elution of antibodies by arginine and arginine derivatives in affinity column chromatography.

It has been shown that the recovery of monomeric antibodies from protein A affinity chromatography is enhanced significantly by using arginine as an eluent. To extend the applications of arginine to antibody purification and obtain an insight into the mechanism of arginine elution, we compared arginine with citrate, guanidine hydrochloride (GdnHCl), arginine derivatives, and other amino acids in protein A chromatography. We also applied arginine to elution of polyclonal antibodies (pAbs) in antigen affinity chromatography. As described previously, arginine was effective in eluting monoclonal antibodies IgG1 and IgG4. Two arginine derivatives, acetyl-arginine and agmatine, resulted in efficient elution at pH 4.0 or higher, and this was comparable to arginine. On the other hand, other amino acids, such as glycine, proline, lysine, and histidine, are much less effective than arginine under identical pH conditions. Whereas elution increased with arginine concentration, elution with citrate was insignificant in excess of 1 M at pH 4.3. Arginine was also effective in fractionation of pAbs using antigen-conjugated affinity columns. Although GdnHCl was also effective under similar conditions, the eluted material showed more aggregation than did the protein eluted by arginine.

Animals↗

PubMed2005

Arginine as an effective additive in gel permeation chromatography.

A major problem in gel permeation chromatography (GPC) or size exclusion chromatography is non-specific binding of applied proteins to the column matrix (stationary phase). We have tested an aqueous arginine solution as the GPC mobile phase on silica-based and polymer-based columns, using mouse monoclonal antibody and recombinant human activin, interleukin-6, basic fibroblast growth factor, and interferon-gamma as model proteins. We observed that addition of arginine to the mobile phase improves separation of the proteins and their soluble aggregates from the GPC columns, which suggests that arginine is an effective additive for the GPC mobile phase.

Activins↗

PubMed2005

A novel "reverse screening" to identify refolding additives for activin-A.

A general approach for refolding recombinant proteins from inclusion bodies (IBs) is to screen conditions, that facilitate a conversion of unfolded to folded structure and minimize a conversion of unfolded to misfolded and aggregated structures. In this simplified model, such conditions may be those that stabilize the native protein and/or reduce aggregation. In this paper, a novel screening approach, termed reverse screening, was developed using a native activin. Activin-A, a member of transforming growth factor beta superfamily, is a homodimeric protein with nine disulfide bonds. We examined partial unfolding process of native activin-A dissolved in a buffer containing moderate concentrations of denaturant and reducing reagent (i.e., 1.5 M urea and 0.2 mM dithiothreitol). The recovery of the protein was followed by reverse-phase high performance chromatography analysis. Without additives, activin-A showed about 60% loss of the protein due to aggregation after 12-h incubation in the above condition. We then tested various additives for their effects on the recovery after partial unfolding. One of these additives, sodium taurodeoxycholate (TDCA), greatly increased recovery and suppressed aggregation of the protein. These additives were then tested for refolding activin-A from IBs. TDCA among others is proved to be a highly effective refolding additive. These results strongly suggest that reverse screening using native proteins, if available, may be another approach to discovering effective refolding additives.

Activins↗

PubMed2005

Is arginine a protein-denaturant?

Arginine is a useful solvent additive for many applications, including refolding and solubilization of proteins from insoluble pellets, and suppression of protein aggregation and non-specific adsorption during formulation and purification. However, there is a concern that arginine may be a protein-denaturant, which may limit the expansion of its applications. Such concern arises from the facts that arginine decreases melting temperature and perturbs the spectroscopic properties of certain proteins and contains a guanidinium group, which is a critical chemical structure for denaturing activity of guanidine hydrochloride. Here, we show that although arginine does lower the melting temperatures of certain proteins, the extent is insufficient to cause denaturation of proteins at or below room temperature. The proteins described here show enzymatic activity and folded structure in the presence of arginine, although the local structure around aromatic amino acids is perturbed by arginine. Arginine differs from guandinine hydrochloride in the mode of interactions with proteins, which may be a primary reason why arginine is not a protein-denaturant.

Amino Acids, Aromatic↗

PubMed2005

Nondenaturing solubilization of beta2 microglobulin from inclusion bodies by L-arginine.

Expression of beta2 microglobulin (beta2m) in Escherichia coli resulted in formation of inclusion bodies. Attenuated total reflectance Fourier transform infrared analysis suggested a native-like secondary structure of beta2m in the inclusion bodies. Nondenaturing solubilization of the native-like beta2m from inclusion bodies was achieved using L-arginine solution, which enables an efficient recovery of beta2m with little aggregation. Greater beta2m solubilization from inclusion bodies was obtained at higher temperatures. Low-temperature solubilization yielded beta2m with fluorescence properties identical to those of native beta2m, but its secondary structure was slightly nonnative. Solubilization at moderate temperature gave beta2m with an apparently native structure. We propose an efficient nondenaturing solubilization method combining L-arginine and moderate temperature.

Arginine↗

PubMed2005

Review: Why is arginine effective in suppressing aggregation?

Arginine is finding a wide range of applications in production of proteins. Arginine has been used for many years to assist protein refolding. This effect was ascribed to aggregation suppression by arginine of folding intermediates during protein refolding. Recently, we have observed that arginine facilitates elution of antibodies during Protein-A chromatography and solubilizes insoluble proteins from inclusion bodies, which both can be ascribed to weakening of protein-protein interactions. In order to gain understanding on why arginine is effective in reducing protein-protein interactions and suppressing aggregation, the effects of arginine on stability and solubility of pure proteins have been examined, which showed that arginine is not a protein-stabilizer, but is an aggregation suppressor. However, there is no explanation proposed so far on why arginine suppresses aggregation of proteins. This review addresses such question and then attempts to show differences between arginine and strong denaturants, which are also known as an aggregation suppressor.

Amino Acids↗