Takaharu Sakiyama

On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenomenon

Adsorption of proteins on solid surfaces and their interaction are major concerns in a number of fields such as biology, medicine, biotechnology and food processing, and play an important role from various points of view. Based on practical viewpoints, information on the conformation of the adsorbed protein as well as adsorption characteristics is essential for a systems performance. Although there are still many problems to be solved, extensive studies in recent years, owing to the development in instrumentation and instrumental techniques, reveal the adsorption behavior of proteins in detail. Here, we stress the importance and interesting aspect of protein adsorption on solid surfaces by reviewing findings that have been obtained in recent years.

Recent advances in controlled immobilization of proteins onto the surface of the solid substrate and its possible application to proteomics

Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate- and microarray-based techniques have been developed by a number of re- searchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while re- taining structure and native biological function. In this review, we focus on recent advances in immobilization methodol- ogy for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library sys- tems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

Effects of bienzyme complex formation of cysteine synthetase from escherichia coli on some properties and kinetics.

Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATΔC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168-179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATΔC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATΔC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.

Screening and Characterization of Affinity Peptide Tags Specific to Polystyrene Supports for the Orientated Immobilization of Proteins

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS‐tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site‐specific immobilization of proteins. The various PS‐tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant‐type glutathione S‐transferases (GSTs) fused with the selected PS‐tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C‐terminus, GST‐PS19 and GST‐PS23, when adsorbed on the PS latex beads had a higher affinity than the wild‐type GST, and the specific remaining activity of the immobilized mutant‐type GSTs was approximately 10 times higher than that of the wild‐type GST. The signal intensity detected for GST‐PS19 and GST‐PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti‐peptide antibody specific for the N‐terminus peptide of GST was much higher than that for the wild‐type GST. These findings indicate that the mutant‐type GSTs fused with the selected peptide tags, PS19 and PS23, could be site‐specifically immobilized on the surface of polystyrene with their N‐terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme‐linked immunosorbent assay (ELISA) systems and protein‐based biochips.

Polyelectrolyte complex gel with high pH-sensitivity prepared from dextran sulfate and chitosan

Swelling equilibria of polyelectrolyte complex gels composed of dextran sulfate and chitosan were studied in dilute NaOH or HCl solutions of various pH values with or without NaCl. The complex gel with approximately equal concentrations of sulfate and amino groups was found to be highly sensitive to the change in external pH in a narrow alkaline range. The maximum equilibrium volume of the complex gel was observed at pH 10.5 and was about 300 times as large as the initial one. Compared with the swelling equilibria of a complex gel prepared from κ-carrageenan and chitosan with approximately equal concentrations of sulfate and amino groups, it is suggested that the initial high density of the ionizable functional groups as well as the flexibility of acidic polymer chain contributes to the high pH sensitivity. The increase in ionic strength of the external solution caused the decrease in gel volume and the shift of the pH value at which the maximum swelling occurred. The shift of the pH value was shown to be understood in terms of the change in the internal pH.

Characteristics of serine acetyltransferase from Escherichia coli deleting different lengths of amino acid residues from the C-terminus.

Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10-25 amino acid residues from the C-terminus (SATΔC10-ΔC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATΔC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATΔC10 was inactivated on cooling to 0°C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-Ile mutant SAT as well as SATΔC14, SATΔC20, and SATΔC25 were stable. Since SATΔC10, SATΔC14, and SATΔC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATΔC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.

Adsorption characteristics of tryptic fragments of bovine β- lactoglobulin on a stainless steel surface

As a strategy for the analysis of the mode of protein adsorption onto stainless steel surfaces, peptides obtained by tryptic digestion of bovine beta-lactoglobulin were subjected to adsorption experiments after identification of their primary structures. In the presence of 1 mM KOH, the peptides were scarcely adsorbed onto the surfaces of stainless steel particles from the peptide mixture. The adsorption experiments on isolated peptides showed that the affinities of the peptides for stainless steel surfaces in the presence of 1 mM HNO3 were significantly different from each other. Peptides without any acidic amino acid residues were scarcely adsorbed onto the surface, whereas some peptides with acidic amino acid residues were found to be irreversibly adsorbed onto the surfaces in the acidic pH region. As for the latter peptides, the amount adsorbed on the surface increased with increasing ionic strength. These results indicated that the carboxyl groups on the side chains of the peptides play an important role in the adsorption. Furthermore, the adsorption behavior of beta-lactoglobulin itself was found to be very similar to that of one of the latter peptides.

Contribution of acidic amino residues to the adsorption of peptides onto a stainless steel surface.

The role of the acidic amino acid residues in the adsorption of peptides/proteins onto stainless steel particles was investigated using a peptide fragment from bovine beta-lactoglobulin, Thr-Pro-Glu-Val-Asp-Asp-Glu-Ala-Leu-Glu-Lys (T5 peptide), which has a high affinity to a stainless steel surface at acidic pHs, and its mutant peptides substituted with different numbers of acidic amino acid residues. The adsorption behavior of the mutant peptides as well as the T5 peptide were studied at pH 3 with respect to concentration and ionic strength dependencies and the reversibility of the adsorption process. The behavior of the peptides was generally characterized as two distinct irreversible adsorption modes, Mode I and Mode II. In Mode I, the amounts adsorbed lay on the ordinate at zero equilibrium concentration in the solution, while in Mode II, the amount adsorbed increased with increased equilibrium concentration. The area occupied by the peptides was predicted by molecular mechanics and molecular dynamics. The state of the peptides, when adsorbed, was investigated using FT-IR analysis. The FT-IR analyses revealed that the side carboxylic groups of the peptides adsorbed on the stainless steel surface were ionized, while they were unionized in the solution at pH 3. Thus, the interactions between the carboxylic groups of the peptide and the stainless steel surface can be considered to be largely electrostatic. The peptide having four acidic amino acid residues took a maximum adsorbed amount, the reason for which is discussed.

Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex.

Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0°C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0°C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by α-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.

Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484

Abstract Membrane-surface liquid culture (MSLC) was applied to the production of kojic acid using Aspergillus oryzae NRRL484. The characteristics of kojic acid fermentation by MSLC were compared particularly with those by shaking culture. The maximum concentration of kojic acid produced and the production rate of kojic acid by the MSLC were usually higher than those by the shaking culture. In the shaking culture, the kojic acid production was the highest with 0.05–0.25% yeast extract and the maximum concentration was around 20 mg/ml. In the MSLC, the highest kojic acid concentration of about 30 mg/ml was obtained with 0.25–0.5% yeast extract. By addition of powdered glucose at a final concentration of 10%, 2–3 times at appropriate intervals during the batch MSLC, the concentration of kojic acid increased to over 100 mg/ml and kojic acid crystals formed in the medium. Repeated fed-batch production of kojic acid by MSLC was quite successful. The concentration of kojic acid produced in each batch was maintained at 75 mg/ml or more with a yield of around 50% for 10 batches and 75 d when the medium contained 0.25% yeast extract. The kojic acid productivity by the repeated fed-batch MSLC was 14.2 g/l/d, about 6 times higher than that by the shaking culture with the medium containing 0.25% yeast extract. After the 11th batch, the production rate decreased, probably due to an increased amount of cells formed on the membrane.