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Summary Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost‐effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost‐effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose‐binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin‐related modifier (SUMO) and SUMO‐specific protease were used to remove it. This method, together with size‐exclusion chromatography, enabled purification of human interleukin‐6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium‐mediated transient expression. Plant‐produced hIL6 (P‐hIL6) contained less than 0.2 EU/μg (0.02 ng/mL) endotoxin. P‐hIL6 activated the Janus kinase‐signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL‐21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.

This paper analyzes the effect of the handset subsidy and the Mobile Number Portability subscriber subsidy regulation, which are the main regulation...

The mapping capability of atomic force microscopy (AFM) enabled us to see captured prostate-specific antigens (PSAs) on a spot microarrayed with the corresponding antibody and count the number of the antigens in a submicrometer area. To enhance the reliability and the reproducibility of the approach, a third-generation dendron was employed for the surface treatment. The specific force between the captured PSA and the detection antibody (5A6) was measured after cross-linking, and the mean unbinding force was 56 +/- 2 pN. At 100 fM, there were 12 captured antigens in 4.32 x 10(4) nm(2), and the number was dependent upon the concentration. A larger hydrodynamic distance (8 +/- 2 nm) of the immunocomplex resulted in a cluster of pixels corresponding to the single complex in a map recorded over a selected area with a positional interval of 3 nm, and this feature helped to discriminate between pixels of the specific interaction and the nonspecific ones. The results indicate that the approach can be applicable to the quantitative analysis of the antigen in a sample and imply that it can be extended to a sample of very low copy numbers as long as the size of the microarrayed spot is reduced.

Vast numbers of studies and developments in the nanotechnology area have been conducted and many nanomaterials have been utilized to detect cancers at early stages. Nanomaterials have unique physical, optical and electrical properties that have proven to be very useful in sensing. Quantum dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, gold nanowires and many other materials have been developed over the years, alongside the discovery of a wide range of biomarkers to lower the detection limit of cancer biomarkers. Proteins, antibody fragments, DNA fragments, and RNA fragments are the base of cancer biomarkers and have been used as targets in cancer detection and monitoring. It is highly anticipated that in the near future, we might be able to detect cancer at a very early stage, providing a much higher chance of treatment.

A polystyrene microtiter plate was coated with a molecular layer of a cone-shaped dendron as a means of providing proper spacing between immobilized biomolecules. For the coating preparation, di(ethylene glycol) vinyl ether was grafted onto the surface of the microtiter plate by a plasma process followed by self-assembly of a second-generation dendron (9-acid) or a third-generation dendron (27-acid). Contact angle analysis revealed a pronounced increase in the hydrophilicity upon plasma grafting, while the hydrophilicity reverted/decreased after dendron immobilization. For analysis by force-based atomic force microscopy (AFM), oligonucleotides were immobilized onto the AFM tip and the plate. The DNA-DNA interaction was observed at all spots examined, which implied that coating of the dendrons was uniform over the entire surface. The effectiveness for biomolecular assays of the spacing on dendron-modified microtiter plates was examined by carrying out an enzyme-linked immunosorbent assay (ELISA), where enhanced detection of different fragments of amyloid beta protein (A beta) was observed when compared with other conventional plates, such as untreated polystyrene or maleic anhydride activated plates. The positive influence of the mesospacing between biomolecules on the microtiter plates for this assay was confirmed.

A peptide chAbeta30-16 (15-mer; CTFVRTHIFCKEHQF) was designed to bind to a region encompassing the entire polymerization-related (16KLVFF20) and part of the polymerization and toxicity-related (25GSNKGAIIGLM35) regions of amyloid beta-protein, Abeta1-42 by a hydropathic complementary approach. This peptide efficiently binds to Abeta and blocks intermolecular interaction and the formation of Abeta aggregates. In addition, the peptide neutralizes the cell toxicity of Abeta fibrils. The chAbeta30-16 peptide or its derivatives may be a starting point for the future development of drugs that prevent the neurotoxicity and deposition of Abeta in the brain of Alzheimer's disease.

From the cloned heavy and light chains of a murine monoclonal antibody (mAbB23) which is specific for human apolipoprotein (apo) B-100 of plasma low-density lipoproteins, a vector was designed for expression of a single-chain antibody (scFv) of mAbB23 in Escherichia coli . The expression vector was constructed so that the scFv gene (V L -linker-V H ) was expressed under the control of the T7 promoter. The inclusion body of scFv was isolated from E. coli lysate and solubilized in 6 M guanidine-hydrochloride without reducing agents, followed by refolding through slow dilution into refolding buffer. After complete removal of the remaining denaturant by dialysis, the soluble scFv was purified through an apo B-100-coupled affinity column, and an active fraction, which had an antigen-binding activity comparable with that of native Fab, was easily obtained. The expression and in vitro refolding of scFv resulted in production of an active molecule in a yield of 15–20 mg per 1-liter flask cultivation.

Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL). In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E. coli. The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E. coli. The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS.

Background: Estrogen replacement therapy in post-menopausal women reduces the risk of coronary artery disease. One of the possible mechanisms of this effect is the modification of lipid profiles. However, there is controversy concerning the effects on lipoprotein(a) [Lp (a)] and lipid levels of progestogens administered with estrogen. Methods: Five hundred fifty-one postmenopausal women were divided into 5 groups: group 1,0.625 mg of conjugated equine estrogen (CEE) (n=140); group 2,0.625 mg of CEE plus 5 mg of medroxyprogesterone acetate (MPA) (n=97); group 3, 0.625 mg of CEE plus 10 mg of MPA (n=109); group 4, 2 mg of estradiol valerate plus 0.5 mg of norgestrel (n=134); and group 5, control (n=71). The Lp(a) and lipid levels were measured before and 2,6, and 12 months after hormone replacement therapy. Results: Estrogen replacement therapy for 12 months lowered the Lp(a) level by 37.1%. The addition of progestogen attenuated the Lp(a)-lowering effect of estrogen. The high-density lipoprotein cholesterol (HDL-C) level was markedly increased in group 1 (16.5%), was moderately increased in groups 2 (10.8%) and 3 (11.3%), and was not changed in group 4. The low-density lipoprotein cholesterol level was decreased by 10.9% to 17.6% in all the treatment groups. Estrogen replacement therapy for 2, 6, and 12 months raised the HDL-C level by 7.2%, 17.4%, and 17.8%, respectively. In the group with combined estradiol plus norgestrel therapy, the HDL-C level was decreased after 2 months and was not changed after 6 and 12 months. The groups that received CEE plus MPA showed intermediate effects between the group that received CEE only and the group that received estradiol plus norgestrel. Conclusions: Combined estrogen and progestogen therapy may have effects on the heart different from those of estrogen therapy alone because of adverse impact of progestogens on Lp(a) and HDL-C levels. The effects of progesterones were dependent on the androgenic potency of progestogen and the duration of therapy. Arch Intern Med. 1996;156:1693-1700

Apolipoprotein (apo) A-I, an abundant protein in high-density lipoproteins (HDL), has received considerable clinical attention as a negative predictor of coronary artery disease, because the plasma concentration of apo A-I has been shown to be inversely correlated with the incidence of coronary artery disease. A direct sandwich enzyme-linked immunosorbent assay (ELISA) for determination of the level of human serum apo A-I was developed using highly purified anti-apo A-I polyclonal antibodies raised in rabbits as the capturing antibody, and as the detecting antibody as well. Microtiter plates were coated with purified anti-apo A-I polyclonal antibodies. The concentration of apo A-I in samples was determined from the peroxidase color reaction. The sensitivity of the assay was determined to be 10 ng of apo A-I per ml of serum. The working range, where the standard curve showed a linearity, was between 10 and 100 ng/ml. The mean intra-assay and inter-assay coefficients of variation (CV) were

Levansucrase gene (levU) ofZ. mobilis had been cloned and overexpressed inEscherichia coli (Song, K.-B. and Rhee, S.-K. (1994)Biotechnol. Lett. 16, 1305–1310). The levansucrase was purified and specific antibodies against it were raised in rabbits. Specificity of the antibodies was demonstrated by Western analysis. By using the antibodies, an efficient, enzyme-linked immunosorbent assay (ELISA) amenable to immunochemical analysis of the levansucrase has been developed. The working range of levansucrase concentration in the developed ELISA was between 75 and 300 ng/ml. The assay was very sensitive to detect the levansucrase as little as 37 ng/ml.

Force-based atomic force microscopy (AFM) was used to detect HCV (hepatitis C virus) RNA directly and to quantitatively analyse it without the need for reverse transcription or amplification. Capture and detection DNA probes were designed. The former was spotted onto a substrate with a conventional microarrayer, and the latter was immobilized on an AFM probe. To control the spacing between the immobilized DNAs on the surface, dendron self-assembly was employed. Force–distance curves showed that the mean force of the specific unbinding events was 32 ± 5 pN, and the hydrodynamic distance of the captured RNA was 30–60 nm. Adhesion force maps were generated with criteria including the mean force value, probability of obtaining the specific curves and hydrodynamic distance. The maps for the samples whose concentrations ranged from 0.76 fM to 6.0 fM showed that cluster number has a linear relationship with RNA concentration, while the difference between the observed number and the calculated one increased at low concentrations. Because the detection limit is expected to be enhanced by a factor of 10 000 when a spot of 1 micron diameter is employed, it is believed that HCV RNA of a few copy numbers can be detected by the use of AFM.

We have cloned and constructed plasmid vectors, pETB23H and pETB23L, for bacterial expression of heavy (H) and light (L) chain cDNAs of Fab' of mAbB23 a monoclonal antibody specific to human plasma apolipoprotein (apo) B-100. The H- and L-chains were expressed as insoluble inclusion bodies in the cytoplasm of Escherichia coli. The inclusion bodies of both chains were isolated from the cell lysate, solubilized in 6 M guanidium-HCl, and mixed in equal molar amounts. Refolding was performed in three stages of dialysis: first, dialysis against 3 M guanidium buffer, next, continuous decrement of guanidium in the dialysis buffer through slow addition of 1 M guanidium buffer, and finally, dialysis against a buffer without guanidium. After the refolding, active Fab' (rFab') was purified through an apo B-100-coupled affinity column. When compared by ELISA, the rFab' had a slightly decreased antigen-binding activity (about 0.7-fold) compared with native Fab. The refolding yield was maximum (75%) when performed at the protein concentrations not more than 0.4 mg ml(-1), whereas the yield decreased exponentially at higher concentrations. The maximum recovery was obtained at the refolding concentration of 1.8 mg ml(-1), where the yield was about 45%. Overall, 2.4-3.0 mg of active rFab' specific to apo B-100 was successfully obtained from 1 l cultivation of E. coli cells.

Production and use of anti-apolipoprotein(a) monoclonal antibodies (MAbs) specific to single copy regions in the polymorphic lipoprotein(a) (Lp(a)) has been emphasized to be important for the standardization of measurements of the coronary heart disease risk factor, Lp(a). Here, mouse MAbs were prepared against the kringle V (V) and protease (P) domains of human apolipoprotein(a) (apo(a)), which domains are present in single copy in the apo(a) molecule. The cDNA for apo(a)VP was cloned from human liver cDNA library, and the V-P recombinant protein overexpressed in Escherichia coli was used as an antigen for the antibody production. Two antibodies named as MAb(a)20 and MAb(a)23 were finally produced, and they were characterized for their binding specificity and epitopes. The specificity of the antibodies was confirmed by an immunoblotting procedure and an enzyme-linked immunoassay (ELISA). It was shown that the antibodies had little, if any, cross-reactivity with human plasminogen, which is relatively abundant in human serum and is highly homologous (85%) with apo(a) in amino acid (aa) sequence. For epitope analysis, 3'-deletional series of apo(a)VP cDNA were constructed, and expression products of them were analyzed for the binding MAb(a)20 and MAb(a)23 do. It has been revealed that distinct epitopes were recognized by the two MAbs: MAb(a)23 (gamma2b, kappa) bound to the V region about 60 aa downstream from the N-terminal, and MAb(a)20 (gamma1, kappa) bound to the P region close to the C-terminal. A one step-sandwich ELISA system for Lp(a) was developed using MAb(a)20 as a capturing antibody and horseradish peroxidase (HRP)-coupled MAb(a)23 as a detecting antibody. The assay was found to be sensitive and useful for detecting Lp(a) in the range of 4-150 microg/dL (80 pM-3 nM).

An active form of single-chain antibody (scFv) has been produced in Escherichia coli for murine monoclonal antibody MabA34 (γ1, κ), which is specific for human plasma apolipoprotein (apo) A-I. The complementary DNAs (cDNAs) encoding the variable regions of heavy chain (V H ) and light chain (V L ) were connected by a (Gly 4 Ser) 3 linker using an assembly polymerase chain reaction. The construct (V L -linker-V H ) was placed under the control of highly efficient T7 promoter system. The cloned scFv was expressed in E. coli as inclusion bodies. After purification from E. coli lysate using sonication and low speed centrifugation, the inclusion body was solubilized and denatured in the presence of 8 M urea, renatured by dialysis, and scFv was finally purified using antigen-affinity chromatography. The purity and activity of purified scFv were confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), Western blotting and enzyme-linked immunosorbent assay (ELISA). The affinity constant was determined by a biosensor method using the BIAcore system. The results showed that the yield of correctly refolded scFv was more than 20 mg l −1 of E. coli flask culture and the specific binding activity to apo A-I was retained with an affinity constant of 6.74×10 −8 M ( K d ). A notable thing is that guanidine–HCl as a denaturant induced more multimeric formation in the subsequent refolding procedure for the scFv of MabA34 and thus, it was not suitable as urea was. This fact is uncommon for what is generally known for the denaturation and refolding of recombinant antibodies.

We have determined the nucleotide (nt) sequences encoding the heavy (H)- and light (L)-chains of the Fab fragment of a murine monoclonal antibody, MabA34 (gamma1, kappa), which is specific for human plasma apolipoprotein A-I of high-density lipoproteins. The variable (V) regions of the H- and L-chains were revealed to be members of mouse H-chain subgroup II(A) and kappa L-chain subgroup II, respectively. A few unusual amino acids in the V region of the H-chain, and nt residues probably introduced by somatic mutations from germline genes were also identified.

We have determined the nucleotide (nt) sequences encoding the heavy (H)- and light (L)-chains of the Fab fragment of a murine monoclonal antibody, MabA34 (γ,κ), which is specific for human plasma apolipoprotein A-I of high-density lipoproteins. The variable (V) regions of the H- and L-chains were revealed to be members of mouse H-chain subgroup II(A) and κ L-chain subgroup II, respectively. A few unusual amino acids in the V region of the H-chain, and nt residues probably introduced by somatic mutations from germline genes were also identified.

Objective: To investigate the serial changes in Lp(a) lipoprotein levels with the loss of female sex hormones by surgical menopause and with estrogen replacement therapy in the same women. Patients and Methods: Forty-four premenopausal women who underwent a transabdominal hysterectomy (TAH) because of benign gynecological disorders were divided into two groups: women who underwent a TAH and unilateral salpingo-oophorectomy (n=31) and women who underwent a TAH and bilateral salpingo-oophorectomy (n=13). In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, 0.625 mg of conjugated equine estrogen was given daily 2 months after the operation. The levels of Lp(a) lipoprotein and lipids were measured before and at 2 and 4 months after the operation. Results: In the group of women who underwent a TAH and bilateral salpingo-oophorectomy, the mean (±SD) concentration of Lp(a) lipoprotein was increased by 24.5% from 0.48±0.47 mmol/L (18.4± 18.3 mg/dL) to 0.59±0.54 mmol/L (22.9±21.0 mg/dL) after 2 months ( P P P Conclusions: The Lp(a) lipoprotein levels appear to be closely associated with female sex hormones. This association might play a pivotal role in postmenopausal increases of atherosclerotic diseases and cardioprotective effect of estrogen in postmenopausal women. (Arch Intern Med. 1996;156:500-504)

A method convenient for isolation of DNA sequences capable of directing gene transcription in both organisms of E. coli and yeast is described. The method is composed of sequential steps of phenotypic selection for chloramphenicol resistance, first in E. coli and then in yeast. A series of promoter-probe, shuttle plasmid vectors between yeast and E. coli were constructed and utilized in the method.