Tandem multimer expression of angiotensin I-converting enzyme inhibitory peptide in Escherichia coli.

It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion.

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).

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.

Efficient production of single-chain Fv antibody possessing rare codon linkers in fed-batch fermentation.

Single-chain Fv antibody (scFv) having 2 types of polypeptide linkers with or without rare codons, namely scFv (G(4)S)(3)(R) and scFv No.10 (with rare codons) and scFv (G(4)S)(3) and scFv No.10(NR) (without rare codon), were expressed under controllable conditions in batch and fed-batch fermentation, in order to compare volumetric productivity and specific productivity levels of scFvs as a soluble form.

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.

Preparation of monodisperse and size-controlled poly(ethylene glycol) hydrogel nanoparticles using liposome templates.

Liposomes were used as templates to prepare size-controlled and monodisperse poly(ethylene glycol) (PEG) hydrogel nanoparticles. The procedure for the preparation of PEG nanoparticles using liposomes consists of encapsulation of photopolymerizable PEG hydrogel solution into the cavity of the liposomes, extrusion through a membrane with a specific pore size, and photopolymerization of the contents inside the liposomes by UV irradiation. The size distributions of the prepared particles were 1.

Angiotensin-I converting enzyme (ACE) inhibitory mechanism of tripeptides containing aromatic residues.

Angiotensin-I converting enzyme (ACE) plays important roles in the regulation of blood pressure, and ACE inhibitory peptides in food materials have attracted attention for their antihypertensive function. In this study, the function of amino acids in ACE inhibitory tripeptides was clarified.

High efficiency production of astaxanthin in an airlift photobioreactor.

In photobioreactors, photosynthetic microorganisms are exposed to certain light/dark cycles caused by light intensity distribution and mixing inside the photobioreactor. In this study, Haematococcus pluvialis was cultivated in an airlift and a bubble column photobioreactor, and the cell growth and astaxanthin production were compared to clarify the effects of liquid circulation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

High performance in protease refolding by application of a continuous system using immobilized inhibitor.

In the reoxidative refolding of Streptomyces griseus trypsin, which is a serine protease having three S-S bonds per molecule, a synthetic inhibitor immobilized on agarose beads was applied in order to avoid the digestion of non-renatured protease molecules by renatured ones. A semi-continuous refolding system was fabricated by packing such inhibitor-immobilized gels in a glass column and the optimal operating conditions were investigated. Taking account of the effective conditions surveyed in the system, a continuous refolding system was constructed and operated to achieve higher performance.

Effects of nutrient supply methods and illumination with blue light emitting diodes (LEDs) on astaxanthin production by Haematococcus pluvialis.

In order to increase the cell concentration and the accumulation of astaxanthin, the effects of nutrient concentration, pH, illumination and methods of supplying nutrients were studied for the cultivation of Haematococcus pluvialis. The replacement of media to avoid the deficiency of nutrients increased the cell concentration above 1 mg-dry cell cm(-3) without induction of astaxanthin accumulation. Illumination with blue light emitting diode lamps and nutrient starvation induced accumulation of astaxanthin, and the interactive effects of these two increased the astaxanthin concentration to 76 mug cm(-3).

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.