Fluorescence-based peptide screening using ligand peptides directly conjugated to a thiolated glass surface.

Functional peptides from peptide libraries are frequently screened using an array format. We report here results of a feasibility study of fluorescence-based peptide screening using an array format on surface-modified glass. The surface of an amine-coated glass slide was modified to contain thiol groups by iminothiolane treatment. The epsilon-amine of the C-terminal lysine from a ligand peptide was iodinated and then spotted onto the thiolated glass surface to covalently conjugate the ligand peptide to the surface via a thioether bond. This covalent immobilization allowed the ligand peptides to withstand washing steps by tightly adhering to the glass surface and confining their subsequent binding reactions within a spotted area. Two representative peptides were used as the ligand peptides; a 'target' (positive) heptapeptide that could specifically bind to trypsin, and a 'control' (negative) hexapeptide that had no binding affinity with trypsin. When fluorescein isothiocyanate-labeled trypsin was reacted with the ligand peptides, the target peptide demonstrated distinctively higher (ca. 8.7-fold) fluorescence intensity that was easily differentiated from the control peptide by a fluorescence scanner. A separate experiment using a quartz crystal microbalance confirmed that the difference in binding mass (ca. 9.1-fold) was very close to that seen in fluorescence intensity. These results suggested a quantitative, 1:1 correlation between mass and fluorescence signals. Furthermore, a smaller spot volume and a higher ligand peptide concentration resulted in higher fluorescence signal intensity. This study provides information on the potential for using fluorescence-based screening of functional peptides on a glass array format.

Effect of light intensity and frequency of flashing light from blue light emitting diodes on astaxanthin production by Haematococcus pluvialis.

Flashing light from blue light emitting diodes is an effective method for the reduction of energy consumption in the bioproduction of astaxanthin by Haematococcus pluvialis. We investigated the effects of light intensity and frequency on the final astaxanthin concentration in bioproduction by H. pluvialis grown mixotrophically. The final astaxanthin concentration under illumination with flashing light, with frequencies ranging from 25 to 200 Hz, was dependent on the light intensity and on the duty cycle and was equivalent, or higher, in comparison with that under illumination with continuous light at the same incident intensity. The light intensity determined the maximum attainable concentration of astaxanthin under continuous illumination. Under illumination with flashing light, the ratio of the final astaxanthin concentration to the maximum concentration at a specific light intensity was correlated to the duty cycle in the frequency range from 25 to 200 Hz. The effect of lower frequencies on enhanced astaxanthin production under flashing light was also studied; at levels as low as 1 Hz, higher final astaxanthin concentrations were observed under flashing light compared to concentrations attained under continuous light.

Proposal for a new categorization of aseptic processing facilities based on risk assessment scores.

Risk assessment of aseptic processing facilities was performed using two published risk assessment tools. Calculated risk scores were compared with experimental test results, including environmental monitoring and media fill run results, in three different types of facilities. The two risk assessment tools used gave a generally similar outcome. However, depending on the tool used, variations were observed in the relative scores between the facilities. For the facility yielding the lowest risk scores, the corresponding experimental test results showed no contamination, indicating that these ordinal testing methods are insufficient to evaluate this kind of facility. A conventional facility having acceptable aseptic processing lines gave relatively high risk scores. The facility showing a rather high risk score demonstrated the usefulness of conventional microbiological test methods. Considering the significant gaps observed in calculated risk scores and in the ordinal microbiological test results between advanced and conventional facilities, we propose a facility categorization based on risk assessment. The most important risk factor in aseptic processing is human intervention. When human intervention is eliminated from the process by advanced hardware design, the aseptic processing facility can be classified into a new risk category that is better suited for assuring sterility based on a new set of criteria rather than on currently used microbiological analysis. To fully benefit from advanced technologies, we propose three risk categories for these aseptic facilities.

Monitoring minimization of grade B environments based on risk assessment using three-dimensional airflow measurements and computer simulation.

A practical, risk-based monitoring approach using the combined data collected from actual experiments and computer simulations was developed for the qualification of an EU GMP Annex 1 Grade B, ISO Class 7 area. This approach can locate and minimize the representative number of sampling points used for microbial contamination risk assessment. We conducted a case study on an aseptic clean room, newly constructed and specifically designed for the use of a restricted access barrier system (RABS). Hotspots were located using three-dimensional airflow analysis based on a previously published empirical measurement method, the three-dimensional airflow analysis. Local mean age of air (LMAA) values were calculated based on computer simulations. Comparable results were found using actual measurements and simulations, demonstrating the potential usefulness of such tools in estimating contamination risks based on the airflow characteristics of a clean room. Intensive microbial monitoring and particle monitoring at the Grade B environmental qualification stage, as well as three-dimensional airflow analysis, were also conducted to reveal contamination hotspots. We found representative hotspots were located at perforated panels covering the air exhausts where the major piston airflows collect in the Grade B room, as well as at any locations within the room that were identified as having stagnant air. However, we also found that the floor surface air around the exit airway of the RABS EU GMP Annex 1 Grade A, ISO Class 5 area was always remarkably clean, possibly due to the immediate sweep of the piston airflow, which prevents dispersed human microbes from falling in a Stokes-type manner on settling plates placed on the floor around the Grade A exit airway. In addition, this airflow is expected to be clean with a significantly low LMAA. Based on these observed results, we propose a simplified daily monitoring program to monitor microbial contamination in Grade B environments. To locate hotspots we propose using a combination of computer simulation, actual airflow measurements, and intensive environmental monitoring at the qualification stage. Thereafter, instead of particle or microbial air monitoring, we recommend the use of microbial surface monitoring at the main air exhaust. These measures would be sufficient to assure the efficiency of the monitoring program, as well as to minimize the number of surface sampling points used in environments surrounding a RABS.

Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface.

Glutathione S-transferase genetically fused with an affinity peptide tag, PS19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS19R10, in which (10)Lys in PS19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immunosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein.

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes.

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.

Optimization of silica-based media for antibody purification by protein A affinity chromatography.

Considering the large molecular size of IgG antibodies widely used for therapeutic applications, the pore size, pore volume and coupling density of silica-based media were optimized for the effective large-scale purification, using an antibody-protein A affinity system. Silica media, with average pore sizes from 70 nm to 140 nm and surface areas of 26-67 m(2)/g, were prepared and coupled with protein A. The static adsorption capacity and dynamic binding capacity of bovine and human IgG were measured at superficial liquid velocities ranging from 94 to 720 cm/h. The volumetric coefficient of mass transfer of the alkali-treated silica-based protein A media, with a pore size of 110 nm, was four times higher than the values for cross-linked agarose media and thus had high dynamic binding capacities at high superficial liquid velocities.

Characteristics of a liposome immunoassay on a poly(methyl methacrylate) surface.

Liposome immunoassay (LIA) is based on enzyme immunoassay (EIA) but the detection sensitivity could be significantly enhanced by using antibody-coupled immunoliposomes encapsulating HRP (horse radish peroxidase). Here, we applied LIA to non-porous poly(methyl methacrylate) (PMMA) and polystyrene (PS) surfaces to compare its detection sensitivity with that of EIA, using rabbit IgG (a ligand molecule) and anti-rabbit IgG antibody (a capture molecule) as the model system. LIA developed much stronger color signals than EIA, especially at a lower concentration range (< ca. 1 microg mL(-1)). PMMA showed higher affinity toward rabbit IgG than the PS surface, and the anti-rabbit IgG antibody adsorbed on PMMA was more stable than that on PS. Furthermore, the effects of spot volume and antibody concentration on the signal density were analyzed. The signal density increased as the antibody concentration increased, but it was not significantly affected by the spot volume (2.5-20 microL). In conclusion, LIA on PMMA as a solid support is a very useful, highly sensitive microarray detection system.

Production of single-chain variable fragment antibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feeding methods.

This study examined the effects of two methods of methanol feeding, DO-stat and methanol concentration control, in fed-batch and continuous cultures of Pichia pastoris on cell growth and single-chain variable fragment antibody (scFv) expression. By maintaining the methanol concentration at 3.9 g l(-1) in fed-batch culture, a scFv concentration of 198 mg l(-1) was obtained. In continuous culture using both methanol feeding methods, the scFv concentration in the fermentation broth increased with a decreasing dilution rate. A maximum scFv concentration of 810 mg l(-1) at a dilution rate of 0.0094 h(-1) was obtained by maintaining the methanol concentration at 3.9 g l(-1). Although the specific methanol consumption rate was the same for both methods, the specific productivity of scFv was higher in methanol concentration control from 0.0094 to 0.049 h(-1) than it was in DO-stat control. Therefore, continuous culture with methanol feeding by the concentration control method shows promise for the industrial scale production of recombinant proteins by Pichia pastoris.

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column.

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.

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.

Degradation of cyanuric acid in soil by Pseudomonas sp. NRRL B-12227 using bioremediation with self-immobilization system.

The rates of degradation of cyanuric acid, a key intermediate in a metabolic pathway of s-triazine herbicides, were measured for Pseudomonas sp. NRRL B-12227. The rate of degradation was affected by the rate of cyanuric acid transport through cell membranes and the activity of cyanuric acid amidohydrolase inside the cells. At low concentrations of cyanuric acid, the acclimation of cells to cyanuric acid and/or added nutrients effectively enhanced the degradation rate. The strain was also applied to bioremediation using a Bioremediation with Self-Immobilization System (BSIS), in which Pseudomonas sp. NRRL B-12227 cells were co-immobilized with Bacillus subtilis, the latter of which secretes a viscous polymer, in a shallow layer of soil packed in a column. More than 70% of the Pseudomonas sp. NRRL B-12227 cells were co-immobilized with the B. subtilis in a 7.5 cm layer of the packed soil by self-aggregation. More than 60% of the 1 mM cyanuric acid supplied to the packed soil was degraded in this layer during a 72 h period.

Effect of flashing light from blue light emitting diodes on cell growth and astaxanthin production of Haematococcus pluvialis.

To conserve energy in the production of astaxanthin by the green alga Haematococcus pluvialis, we utilized intermittent flashing light from blue light emitting diodes (LEDs) and investigated the effects of the incident light intensity (2-12 micromol m(-2) s(-1)), duty cycle (17-67%) and frequency (25-200 Hz) of flashing on the cell growth and astaxanthin production. In the above ranges, the final astaxanthin concentration under illumination by flashing light was significantly higher than that obtained under illumination with continuous light at the same incident intensity. For example, flashing light at an incident intensity of 8 micromol m(-2) s(-1) gave the same final astaxanthin concentration that was obtained under continuous light illumination at 12 micromol m(-2) s(-1), thus reducing energy consumption by 1/3. We therefore conclude that flashing light from blue LEDs is a promising illumination method for indoor algal cultivation using photobioreactors.

Effective utilization of transmitted light for astaxanthin production by Haematococcus pluvialis.

A new cultivation method, in which physiological responses of Haematococcus pluvialis to different intensities and wavelengths of illuminating light are utilized, is proposed. In this method, light transmitted through a cultivation vessel illuminated from the opposite side was utilized for the early-phase cultivation of H. pluvialis in another inoculated vessel that was located behind the cultivation vessel, to save time required for the growth of cells. After harvesting cells from the front vessel, the vessel that was originally behind was shifted to the position of the front vessel. The abrupt increase in light intensity caused by shift of the position induced the effective accumulation of astaxanthin. These procedures for inoculation, shift of vessels and harvest were repeated using two vessels arranged in series along the light path. After four repeated cycles of cultivation (840 h from the start of cultivation), using 2.5 cm thick vessels, astaxanthin production per unit volume and the astaxanthin content were higher than obtained in a batch cultivation with two 2.5 cm vessels.

Effects of methanol feeding methods on chimeric alpha-amylase expression in continuous culture of Pichia pastoris.

The effects of two types of methanol feeding methods in a continuous culture of Pichia system on the cell growth and recombinant protein expression were studied using chimeric alpha-amylase as a model protein. With the feeding of methanol by a DO-stat method, the alpha-amylase concentration in the fermentation broth increased with decreasing dilution rate and reached 173 mg/l at a dilution rate of 0.013 h(-1), at which the maximum volumetric productivity of alpha-amylase was obtained. Although almost the same productivity was attained at 0.04 h(-1) with continuous methanol feeding, the alpha-amylase concentration was one third that compared with feeding by the DO-stat method, that is, 55 mg/l. Furthermore, at this dilution rate, the medium volume needed per unit time was three times that required when DO-stat was used. Therefore, continuous culture with methanol feeding by the DO-stat method may be a promising method for the production of recombinant proteins on an industrial scale by Pichia pastoris.

Application of protein-coupled liposomes to effective affinity screening from phage library.

For effective screening by biopanning, we propose a new affinity screening method utilizing protein-coupled liposomes (proteoliposomes) as adsorbents. With multilamellar vesicles (MLVs) composed of dipalmitoylphosphatidylcholine (DPPC): dicetylphosphate (DCP) = 10: 1 (molar ratio), adsorption of nonspecific phage VCSM13 to the liposomes without any blocking was comparable to that on polystyrene tube wall coated with blocking protein. Phages displaying octapeptides specific to an anti-peptide antibody against a peptide antigen (FVNQHLCK) were screened from an octapeptide-displayed phage library by biopanning utilizing liposomes coupled with the antibody (AB-MLVs) or a conventional immunotube coated with the antibody (AB-tube). After four rounds of biopanning, all selected phages displayed homological peptides to the antigen peptide by use of AB-MLVs, while only 15% of the selected phages displayed homological peptides in the conventional biopanning. The octapeptide selected by AB-MLVs against the anti-peptide antibody showed comparable binding affinity, which were determined by the competitive ELISA and an immunoaffinity chromatography, to that of the peptide antigen. Thus, protein-coupled liposomes are useful as adsorbents for screening from combinatorial phage libraries.

Improvement of growth stability of photosynthetic bacterium Rhodobacter capsulatus.

Using a semicontinuous culture method, in which operational parameters such as cell concentration and light intensity distribution were maintained almost constant, instability of the specific growth rate of Rhodobacter capsulatus B-100, a purple bacterium, was observed to be similar to that of R. capsulatus ST-410 when cultivated under high ratios of light intensity on the illuminated side to that of the transmitted light. Such instability was not observed in the cultivation of Chlorella vulgaris, a eukaryotic green alga, even at higher cell concentrations. Under the same conditions, the increase in only the ferrous concentration from 43 microM, the concentration in the original RCV medium, to 172 microM sustained a stable growth, whereas Fe(2+) was slightly consumed during the cultivation. Supplemental illumination with a fluorescent lamp on the transmitted side of a flat plate photobioreactor sustained a moderate level of stable growth, while a halogen lamp slightly affected the growth stability. Our results showed that an increase in Fe(2+) concentration or supplemental illumination improves the growth stability of R. capsulatus.

Fed-batch culture under illumination with blue light emitting diodes (LEDs) for astaxanthin production by Haematococcus pluvialis.

To increase the cell concentration and the accumulation of astaxanthin, the effects of the fed-batch addition of 10-fold-concentrated medium to supply nutrients, as well as illumination with blue light emitting diodes (LEDs), on cell growth and accumulation of astaxanthin were studied for the cultivation of Haematococcus pluvialis. Using the fed-batch addition method, the cell concentration increased above 1 mg-dry cell/cm3, and under illumination with blue LEDs, the astaxanthin concentration reached approximately 70 microg/cm3. This method was much simpler to operate than the medium replacement method in operation and enabled us to attain a higher total yield of astaxanthin.

Effects of light intensity distribution on growth of Rhodobacter capsulatus.

For cultivation of photosynthetic cells under defined light intensity distributions, the repeated batch culture, in which a part of culture broth containing grown cells was repeatedly replaced at predetermined time intervals with a fresh medium to keep the cell concentration constant at an initial value, was employed. By use of this method the effects of the light intensity distribution on the growth characteristics of Rhodobacter capsulatus were studied. Unexpected decreases in the specific growth rate were observed in culture of R. capsulatus at high cell concentrations and a long light path length. Big differences in the light intensities of lightly and darkly illuminated portions in photobioreactors, which reflects the light intensity distribution, seemed to cause this phenomenon, which must be taken into consideration for stable growth of photosynthetic cells.

Characteristics of microblotting assay using immunoliposomes.

To clarify the applicability of a liposome immunoblotting assay to microassay systems, the effects of the sample volume blotted on polyvinylidiene fluoride (PVDF) membrane on the sensitivity and signal intensity were studied and compared with those in a conventional immunoblotting assay utilizing enzyme-labeled antibody. In the liposome immunoblotting assay, signal intensities per unit area (signal density) were almost the same or increased slightly with decreasing blotted sample volume (from 20 to 2 microl) at the same concentration of analyte (human IgM) adsorbed on PVDF membrane. A substrate, 4-chloro-1-naphtol, for color development formed colored precipitates inside the liposomes. Thus, the colored signal was detected without loss of intensity. On the other hand, in the conventional immunoblotting assay, signal densities decreased with decreasing sample volume at the same concentration of analyte. This is caused by partial losses of the colored signal into the substrate solution by diffusion, because the fraction of loss increases with decreasing sample volume. These results show that the liposome immunoblotting assay is a promising method for the detection of analytes blotted in small areas, because its sensitivity is higher than that of the conventional blotting assay.