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A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli . With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His 6 -Ub-hGH), glutathione S -transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and Western blots.

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His6-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinkerTM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His6-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.

In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (μCP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene ofStreptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of μμCP and cSA-fused proteins, is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications, one such being the use in protein-protein assays.

We have isolated putative esterase genes from various bacterial chromosomes. Thirty open reading frames predicted to encode esterases were randomly selected from 13 sequenced bacterial chromosomes and were cloned into an expression vector. The esterase activity of the resulting clones was tested on a tributyrin plate at different pH values and temperatures. Nine out of thirty tested clones exhibited significant tributyrin hydrolyzing activity. The enzyme S5 from the gene b0494 of Escherichia coli, the enzyme S12 from the gene STM0506 of Salmonella typhimurium, and the enzyme S28 from the gene AF1716 of Archaeoglobus fulgidus exhibited high activity at an alkaline pH range. The esterase S11 encoded by the gene PA3859 of Pseudomonas aeruginosa PAO1 and the esterase S21 from the gene SMc01033 of Sinorhizobium meliloti 1021, both showed a sharp increase in enzyme activity above pH 8.0. Furthermore, the enzymes S5, S12, S21, and S28 retained the esterase activity when they were incubated at 50 degrees C, suggesting that these enzymes are thermostable. Subsequent pH vs. activity and temperature vs. activity experiments with selected enzymes in a solution assay system confirmed the validity of the above data. The genome-wide exploration strategy of proteins provided valuable information on the esterases by revealing subtle biochemical differences between the esterases of different sources.

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli . The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.

A newly isolated gene from Pseudomonas sp. KCTC 10122BP, encoding an esterase with enantioselectivity towards racemic ketoprofen (rac-ketoprofen) ethyl ester, was cloned in Escherichia coli and its nucleotide sequence determined. The deduced amino acid sequence predicted an open reading frame (ORF) encoding a polypeptide of 381 amino acid residues (1143 nucleotides) with a calculated isoelectric point of pH 5.32 and molecular mass of 41,149 Da. The primary structure of the enzyme exhibited a significant level of homology (>31%) with those of related enzymes from various sources and an extreme homology (>81%) with five esterases from the genus Pseudomonas. The enzyme was expressed at a high level in an active form in the soluble fraction and purified to homogeneity by a successive chromatographic procedure. The purified enzyme was determined to be a monomer, plus it exhibited a strict selectivity (>99%) and high activity (2360 units/mg-protein) towards (S)-ketoprofen ethyl ester.

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the Saccharomyces cerevisiae -derived prepro α-factor leader in the methylotrophic yeast Hansenula polymorpha . The recombinant hEGF(1–53), when secreted by H. polymorpha , rapidly cleaved to hEGF(1–52) by carboxy-terminal proteolysis, resulting in the accumulation of C-terminal-truncated hEGF(1–52) in the culture medium. To solve this problem, we constructed a H. polymorpha mutant in which the KEX1 gene coding for carboxypeptidase yscα was disrupted. The extent of C-terminal proteolysis of hEGF was significantly reduced when this kex1 disruptant was used as a host strain. After 24 h of shake-flask culture, most of the hEGF secreted by the kex1 disruptant remained intact, whereas more than 90% of the hEGF secreted by the wild-type was C-terminally cleaved. The recombinant hEGF was purified to >98% purity by two sequential steps of preparative scale anion exchange chromatography and reverse-phase HPLC. The authenticity of purified hEGF was confirmed by HPLC, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses.

The near-infrared (NIR) fluorescence probe has better tissue penetration and lower autofluorescence. Indocyanine green (ICG) is an NIR organic dye for extensive biological application, and it has been clinically approved for human medical imaging and diagnosis. However, application of this dye is limited by its numerous disadvantageous properties in aqueous solution, including its concentration-dependent aggregation, poor aqueous stability in vitro, and low quantum yield. Its use in molecular imaging probes is limited because it loses fluorescence after binding to nonspecific plasma proteins, leading to rapid elimination from the body with a half-life of 2 – 4 min. In this study, the multifunctional perfluorocarbon (PFC)/ICG nanoemulsions were investigated with the aim of overcoming these limitations. The PFC/ICG nanoemulsions as a new type of delivery vehicle for contrast agents have both NIR optical imaging and 19 F-MR imaging moieties. These nanoemulsions exhibited less aggregation, increased fluorescence intensity, long-term stability, and physicochemical stability against external light and temperature compared to free aqueous ICG. Also, the PFC/ICG bimodal nanoemulsions allow excellent detection of lymph nodes in vivo through NIR optical imaging and 19 F-MR imaging. This result showed the suitability of the proposed nanoemulsions for non-invasive lymph node mapping as they enable long-time detection of lymph nodes. Electronic supplementary material The online version of this article (doi:10.1186/s40580-014-0006-6) contains supplementary material, which is available to authorized users.

The effective targeting of cancer cell surface antigens is an attractive approach in cancer diagnosis and therapy. Multifunctional nanoprobes with cell-targeting specificity are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. In this study, we have fabricated biocompatible perfluorocan/quantum dot nanoemulsions as bimodal imaging nanoprobes for the targeting of breast cancer cells. Perfluorocarbon/quantum dot nanoemulsions conjugated with monoclonal antibodies, as a type of bimodal imaging nanoprobe based on 19 F-MR and optical imaging, have been synthesized and applied for targeted imaging of three different breast cancer cells (SKBR3, MCF-7, MDA-MB 468), respectively. We have shown that the cancer-detection capabilities of antibody-conjugated PFC/QDs nanoemulsions could be successfully applied to target of various breast cancer cells. These modified PFC/QDs nanoemulsions were shown to target the cancer cell surface receptors specially. Conjugation of ligands to nanoemulsions targeting over-expressed cell surface receptors is a promising approach for targeted imaging to tumor cells. We further propose that the PFC/QDs nanoemulsions could be used in targeted imaging of breast cancer cells. Electronic supplementary material The online version of this article (doi:10.1186/s40580-014-0023-5) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) are emerging new biomarkers for many human diseases. To fully employ miRNAs as biomarkers for clinical diagnosis, it is most desirable to accurately determine the expression patterns of miRNAs. The optimum miRNA profiling method would feature 1) highest sensitivity with a wide dynamic range for accurate expression patterns, 2) supreme specificity to discriminate single nucleotide polymorphisms (SNPs), and 3) simple sensing processes to minimize measurement variation. Here, an ultra-specific detection method of miRNAs with zeptomole sensitivity is reported by applying bi-temperature hybridizations on single-crystalline plasmonic nanowire interstice (PNI) sensors. This method shows near-perfect accuracy of SNPs and a very low detection limit of 100 am (50 zeptomole) without any amplification or labeling steps. Furthermore, multiplex sensing capability and wide dynamic ranges (100 am-100 pm) of this method allows reliable observation of the expression patterns of miRNAs extracted from human tissues. The PNI sensor offers combination of ultra-specificity and zeptomole sensitivity while requiring two steps of hybridization between short oligonucleotides, which could present the best set of features for optimum miRNA sensing method.

BackgroundAdipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited.Methods and resultsWe applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes.ConclusionsThese selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity.

Levansucrase from Zymomonas mobilis expressed in Escherichia coli was immobilized onto the surface of magnetite. Optimum conditions for the immobilization were, pH 4.0; immobilization reaction time of 30 min and 8 U of enzyme per gram of matrix. Immobilization yield was 75% under optimized conditions. The maximum production yield of levan by the immobilized levansucrase was about 42% on a fructose released basis with sucrose as substrate at 100 g/l. Thermal stability of the levansucrase increased by immobilization procedure. Furthermore, immobilized enzyme showed the less polymerizing activity in levan formation, producing the low-molecular weight (MW) levan. The molecular weight of levan can be controlled by using the immobilized levansucrase in high yield. Immobilized levansucrase retained 61% of the original activity after five repeated uses.

A simulation and experimental study has been carried out on the adaptive optimization of fed-batch culture of yeast. In the simulation study, three genetic algorithms based on different optimization strategies were developed. The performance of those three algorithms were compared with one another and with that of a variational calculus approach. The one that showed the best performance was selected to be used in the subsequent experimental study. To confer an adaptability, an online adaptation (or model update) algorithm was developed and incorporated into the selected optimization algorithm. The resulting adaptive algorithm was experimentally applied to fed-batch cultures of a recombinant yeast producing salmon calcitonin, to maximize the cell mass production. It followed the actual process quite well and gave a much higher value of performance index than the simple genetic algorithm with no adaptability.

An optically pure ( S )-naproxen was produced by two-step acetone-treated Candida rugosa lipase (CRL) through enantioselective hydrolysis of racemic naproxen methyl ester. The two-step acetone-treated CRL was much more enantioselective than the crude CRL towards the hydrolysis of ( R,S )-naproxen methyl ester, yielding an enantiomeric excess (ee p ) of >98% and an enantiomeric ratio ( E ) of >100. In terms of hydrolysis reaction rate and enantioselectivity, the optimal reaction conditions were found to be 37 °C and pH 6.0. The scaled-up production of ( S )-naproxen was performed in a batch reactor containing 200 ml of substrate solution (50 mM MES buffer containing 200 mM ( R,S )-naproxen methyl ester). After 156 h of reaction, 38.4% of the initial ( R,S )-naproxen methyl ester was hydrolyzed, yielding an enantiomeric excess of 98% and an enantiomeric ratio of>100. Finally, ( S )-naproxen was recovered from the reaction mixture with an optical purity of 98% and a recovery yield of 95%.

A recombinant human growth hormone (hGH) was expressed as a secretory product in the yeastSaccharomyces cerevisiae. Three different leader sequences derived from the mating factor α1 (MFα1), inulinase and invertase were used to direct the secretion of hGH into the extracellular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the extracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into the medium by the invertase, MFα1, inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase, MFα1 and inulinase leader sequences, respectively.

A simulation and experimental study on the on-line adaptive optimization of fed-batch yeast fermentation process has been performed using genetic algorithms. In simulation study, genetic algorithms based on three different strategies with no adaptation were developed and applied to obtain the optimal feed rate profiles for several initial conditions. The genetic algorithm showed better or similar results compared with an approach based on maximum principle and singular control theory. To confer adaptability on the optimization algorithm developed, an on-line model parameter estimation strategy based on genetic algorithm was developed and incorporated into it. In experimental study, the resulting adaptive optimization algorithm was applied to fed-batch cultures of a recombinant yeast producing calcitonin. It performed on-line model parameter update and optimal feed rate profile calculation simultaneously. It followed the process quite well and gave a much higher value of performance index (in this particular case, cell mass produced) than the simple genetic algorithm with no adaptability.

The characteristics of levan formation by different preparations of levansucrase (free and immobilized enzyme and toluene-permeabilized whole cells), derived from recombinant levansucrase from Zymomonas mobilis expressed in Escherichia coli, were investigated. The maximal yield of levan by the three preparations were similar and were about 70–80% on a fructose-released basis with sucrose as nutrient at 100 g l−1. Immobilized enzyme and toluene-permeabilized whole cells produced low molecular weight levan (2–3 × 106), as determined by HPLC while high molecular weight levan (>6 × 106) was the major product with the free levansucrase. The size of levan can thus be controlled by immobilized levansucrase and toluene-permeabilized whole cells in high yield.

One of the main challenges in the development of new analytical platforms for ultrasensitive bioaffinity detection is jointly achieving a wide dynamic range in target analyte concentration, especially for approaches that rely on multistep processes as a part of the signal amplification mechanism. In this paper, a new surface-based sandwich assay is introduced for the direct detection of B-type natriuretic peptide (BNP), an important biomarker for cardiac failure, at concentrations ranging from 1 aM to 500 nM. This was achieved using nanoparticle-enhanced surface plasmon resonance (SPR) where a DNA aptamer is immobilized on a chemically modified gold surface in conjunction with the specific adsorption of antiBNP coated gold nanocubes in the presence of the biomarker target. A concentration detection range greater than eleven orders of magnitude was achieved through dynamic control of only the secondary nanoparticle probe concentration. Furthermore, detection at low attomolar concentrations was also achieved in undiluted human serum.

Human lipocortin-I was expressed as a secretory product bySaccharomyces cerevisiae harboring an expression system consisting ofGAL10 promoter, inulinase signal sequence and lipocortin-I terminator. Fed-batch fermentation was carried out to overproduce recombinant human lipocortin-I. The culture medium was desalted and concentrated by ultrafiltration, and then subjected to hydroxyapatite column chromatography. The lipocortin-I was purified to >98% purity by single-step hydroxyapatite column chromatography. However, it was found that the purified lipocortin-I was a proteolytically-cleaved form which was cleaved immediately after the basic amino acid Lys26.

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.

A novel two-step acetone treatment method was developed to improve the enantioselectivity of Candida rugosa lipase (CRL) towards the hydrolysis of (R,S)-ketoprofen ethyl ester. After two-step acetone treatment of crude CRL, the CRL was harvested as precipitates and used for the production of optically pure (S)-ketoprofen. The specific activity of two-step acetone-treated CRL was 2.05 kU/mg protein, which corresponds to a 2-fold increase over that of crude CRL. The two-step acetone-treated CRL was considerably more enantioselective than the crude CRL towards (R,S)-ketoprofen ethyl ester, yielding an enantiomeric excess (% eep) of about 100% and an enantiomeric ratio (E) of >100. The hydrolysis activity of acetone-treated CRL increased with an increase in reaction temperature but the enantiomeric excess was >99% regardless of reaction temperature. The production of optically pure (S)-ketoprofen was performed for 108 h in a scaled-up batch reactor containing 200 g of (R,S)-ketoprofen ethyl ester. Consequently, about 90 g of (S)-ketoprofen with an optical purity of 98% was recovered from the reaction mixture.

The INU genes of Kluyveromyces marxianus encode inulinases which are readily secreted from Saccharomyces cerevisiae into the culture medium. To evaluate the utility of the INU signal peptides for the secretion of heterologous proteins from S. cerevisiae, a variety of expression and secretion vectors were constructed with GAL10 promoter and GAL7 terminator. The coding sequence for human lipocortin-1 (LC1) was inserted in-frame with the INU signal sequences, and then the secretion efficiency and localization of LC1 were investigated in more detail and compared with those when being expressed by the vector with the MFalpha1 leader peptide. The vector systems with INU signal peptides secreted ca. 95% of the total LC1 expressed into the extracellular medium, while the MFalpha1 leader peptide-containing vector resulted in very low secretion efficiency below 10%. In addition, recombinant human interleukin-2 (IL-2) was expressed and secreted with the vector systems with INU signal peptide, and a majority fraction of the human IL-2 expressed was found to be secreted into the extracellular medium as observed in LC1 expression. (c) 1995 John Wiley & Sons, Inc.

Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production. Under this condition, 2.8 g L−1 of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography, refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99.

Fed-batch fermentations were performed to overproduce human parathyroid hormone (hPTH) in a Saccharomyces cerevisiae mutant, deficient in yeast aspartic protease 3 (YAP3), developed to minimize the proteolytic cleavage of hPTH. The secretion of hPTH, which was expressed under the control of GAL10 promoter, was directed by the mating factor α pre-pro leader sequence. The intermittent feeding of medium containing carbon mixtures of galactose and ethanol was the feeding strategy, which resulted in the maximum production of intact hPTH of ∼188 mg/l. Although a yap3 -disrupted mutant was used as a host, most of the secreted intact hPTH was degraded at later stages of fermentation. To minimize the loss of intact hPTH, it is important to terminate the fermentation when the concentration of intact hPTH reaches the maximum.

The carboxypeptidase Y (CPY) propeptide from Saccharomyces cerevisiae was developed as a fusion partner for the efficient expression of small polypeptides in Escherichia coli. Six consecutive histidine residues (6xHis) were fused to the N-terminus of the CPY propeptide for the facilitated purification of fusion proteins using immobilized metal ion affinity chromatography. In addition, a methionine or the pentapeptide (Asp)(4)-Lys linker was inserted at the junction between the CPY propeptide and the target polypeptide to release the target polypeptide by digestion with cyanogen bromide or enterokinase. Therapeutically valuable peptide hormones, such as salmon calcitonin precursor (sCAL-Gly), a fragment of human parathyroid hormone (hPTH(1-34)), and human glucagon were successfully expressed in E. coli as fusion polypeptides with the fusion partner. SDS-PAGE analyses showed that the majority of the expressed fusion sCAL-Gly and fusion hPTH(1-34) were present in the form of inclusion bodies, whereas about 66% of the expressed human glucagon was in a soluble form. Almost complete cleavage of the fusion polypeptides was obtained by digestion with enterokinase. Reverse-phase HPLC analyses showed that the target polypeptides released from the fusion proteins were identical to their native forms.

Human lipocortin-I was very efficiently produced as a secretory product by Saccharomyces cerevisiae harbouring an optimized expression casette containing the GAL10 promoter, inulinase signal sequence and the lipocortin-I terminator. To overproduce lipocortin-I, a fed-batch fermentation was performed. The feed medium contained only glucose as sole carbon source and was first fed for cell growth. A mixture of glucose and galactose was then fed for gene induction in parallel with cell growth. During the gene induction period, a significant amount of lipocortin-I accumulated in the culture medium. However, about 45% of the secreted lipocortin-I existed in a proteolytically-cleaved form, cleaved after the basic amino acid Lys 26 . At the end of the culture, the concentration of total extracellular lipocortin-I (intact form+cleaved form) was about 2.1 g/l, accounting for more than 80% of the total extracellular protein.

We have previously reported (Kim et al., J. Dairy Res., 1999, in press) three novel angiotensin-I-converting enzyme (ACE) inhibitory peptides, Tyr-Pro-Glu-Arg, Tyr-Tyr-Pro-Gln-Ile-Met-Gln-Tyr and Asn-Asn-Val-Met-Leu-Gln-Trp, isolated from the tryptic digest of recombinant human αs1-casein. A series of C-terminal di- and tripeptides from these three peptides have now been synthesized and their ACE inhibitory activities have been measured. Among the peptides tested, the tripeptide Leu-Gln-Trp had the highest ACE inhibitory activity (IC50=3.8 μmol l−1).

Human parathyroid hormone (hPTH) was expressed and secreted in Saccharomyces cerevisiae. In batch fermentations performed at pH = 5.6, 6.5, 7.2 and 7.5, optimal production of hPTH (12.1 mg/l) was obtained at pH 7.2 after 24 h of culture. At pH 5.6, most of secreted hPTH was degraded. Proteolysis of hPTH was significantly decreased by increasing the culture pH.

A novel process using O2-enriched air supply was used to suppress the C-terminal proteolytic degradation of recombinant hirudin (r-hirudin) from Saccharomyces cerevisiae. When dissolved O2 was controlled above 20% saturation level using normal air, inactive forms of C-terminally truncated hirudin were observed in culture broth from 48 h of fermentation. The use of O2-enriched air giving above 40% saturation of dissolved O2 suppressed the proteolytic degradation and hence the formation of truncated forms of inactive r-hirudin until 60 h of fermentation.

The genes encoding pro-carboxypeptidase Ys (pro-CPYs) from Saccharomyces cerevisiae and Hansenula polymorpha were cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was present in the form of inclusion bodies, accounting for about 35% of the total cellular protein. The inclusion bodies solubilized in 6 M guanidinum chloride were renatured by dilution 1:60 into renaturation buffer. Further refolding and activation were followed by the addition of 60 μg ml−1 proteinase K into the diluted solution. The specific activities of in vitro activated S. cerevisiae and H. polymorpha CPYs were found to be similar, representing about 25% of that of native S. cerevisiae CPY.

In batch cultures for L-ornithine production in which dissolved oxygen concentration and pH were closely controlled, time changes of redox potential were observed in connection with the profiles of cell, glucose, and ornithine concentrations. It was found that the redox potential profile had four different phases reflecting the physiological state of the culture and that it was closely related to cell concentration change. Effects of glucose and ornithine on the redox potential were identified in a separate series of experiments. On the basis of the experimental results, a correlation of redox potential to glucose, cell, and ornithine concentrations has been proposed. The proposed correlation can be used for on-line estimation of ornithine concentration from on-line data of redox potential, glucose concentration, and cell concentration.

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.

Different feeding strategies of galactose were employed to improve the production of anticoagulant, hirudin, by fed-batch mode of cultivation from recombinant Saccharomyces cerevisiae. The structural gene coding for hirudin was harboured with GAL10 promoter for controlled expression of hirudin and the MFα 1 signal sequence for secretion into the growth medium. A step-wise feeding of galactose was found as more suitable feeding strategy of galactose which resulted in the final hirudin volumetric productivity of 6,840 μg/l · h, than intermittent, continuous and ethanol controlled feeding of galactose. The final volumetric productivity of hirudin obtained by step-wise feeding of galactose was 3.88 fold higher compared with simple batch fermentation.

Human alpha 1-antitrypsin (alpha 1-AT) is a major serine protease inhibitor in plasma, secreted as a glycoprotein with a complex type of carbohydrate at three asparagine residues. To study glycosylation of heterologous proteins in yeast, we investigated the glycosylation pattern of the human alpha 1-AT secreted in the baker's yeast Saccharomyces cerevisiae and in the methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The partial digestion of the recombinant alpha 1-AT with endoglycosidase H and the expression in the mnn9 deletion mutant of S. cerevisiae showed that the recombinant alpha 1-AT secreted in S. cerevisiae was heterogeneous, consisting of molecules containing core carbohydrates on either two or all three asparagine residues. Besides the core carbohydrates, variable numbers of mannose outer chains were also added to some of the secreted alpha 1-AT. The human alpha 1-AT secreted in both methylotrophic yeasts was also heterogeneous and hypermannosylated as observed in S. cerevisiae, although the overall length of mannose outer chains of alpha 1-AT in the methylotrophic yeasts appeared to be relatively shorter than those of alpha 1-AT in S. cerevisiae. The alpha 1-AT secreted from both methylotrophic yeasts retained its biological activity as an elastase inhibitor comparable to that of alpha 1-AT from S. cerevisiae, suggesting that the different glycosylation profile does not affect the in vitro activity of the protein.

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.

Levan, fructo-oligosaccharides and fructosyl derivatives were formed from sucrose using recombinant levansucrase from Rahnella aquatilis. Levan formation was optimal at 30 °C resulting 57 % of the theoretical yield. The more suitable substrate concentration for levan formation was 200 g sucrose/L. Oligosaccharides was accumulated selectively at high substrate concentration. The increase of levan and oligosaccharides formation was not achieved by adding water-miscible organic solvents. Alkyl fructosides were synthesized from various alcohols as fructosyl acceptors by R. aquatilis levansucrase. © Rapid Science Ltd. 1998

In an attempt to induce the secretion of human granulocyte-colony stimulating factor (hG-CSF) in Escherichia coli , the gene encoding mature hG-CSF was fused to the gene for the pectate lyase B (pelB) signal peptide of Erwinia caratovora , and fed-batch fermentation of the recombinant E. coli was performed. No secretion of hG-CSF and no processing of the signal peptide from the fusion protein was observed. Instead, the fusion protein, pelB-hG-CSF, at a concentration of ca. 1.7 g/ l was produced in an inclusion body form. Interestingly, the biological activity of the purified fusion protein was found to be identical to that of mature hG-CSF, indicating that the pelB signal peptide attached to the N-terminal of hG-CSF had no effect on its biological activity.

Human alpha1 -antitrypsin (alpha1-AT) was expressed and secreted in Saccharomyces cerevisiae carrying the gal1 and reg1-501 mutations under the control of GAL10 promoter. Increasing galactose from 0 to 0.5 g/l increased the extracellular concentration of alpha1-AT, though the secretion efficiency was decreased. The final titre of alpha1-AT secreted and the secretion efficiency were 17.9 mu g/ml and 24.8% respectively, with 0.5 g galactose/l. © Rapid Science Ltd. 1998

To produce levansucrase, a fructosyltransferase enzyme, in a recombinant Escherichia coli harboring the levU gene of Zymomonas mobilis, fermentation strategies were examined in terms of induction methods and plasmid stability. Although the recombinant levansucrase was induced rapidly by IPTG, high instability of the plasmid and formation of inclusion bodies were observed. Segregational instability was aggravated by the excretion of β-lactamase into the culture broth during subculturing, which caused an overgrowth of plasmidfree cells. A combination of methicillin (2, 6-dimethoxyphenyl-penicillin) and ampicillin to provide selective pressure was effective in preventing the growth of plasmid-free cells. The population of plasmid-harboring cells was maintained above 95% of the total cells for more than 100 generations under this condition. In order to replace IPTG, which is toxic and too expensive for use in a large scale, d -lactose was tested and found to be favorable as an alternative inducer.

Fed-batch fermentation was conducted to overproduce human lipocortin-I (LC1) in recombinant Saccharomyces cerevisiae controlled by the GAL10 promoter. To optimize the feeding strategy, various fed-batch culture modes were performed with concentrated feed media containing carbon mixtures of galactose (Gal) and glucose (Glu) at three different concentration ratios (Gal : Glu ratios): 9 : 1, 1 : 1, 1 : 9. The cell growth, expression level of LC1, and the plasmid stability were investigated under these fed-batch culture modes. While both glucose and galactose were being fed to the fermentor, the glucose concentration in the culture broth was kept below 1 g/ l for efficient gene expression. High cell concentrations of greater than 100 g dry cell weight/ l were achieved with these fed-batch culture modes. A significant amount of intact LC1 was found to be secreted into the culture medium, but proteolytically cleaved products (des1-26-LC1) were also observed in the culture medium. The fed-batch fermentation with the feed medium at a Gal : Glu ratio of 1 : 1 resulted in the highest LC1 total (intact LC1 + des1-26-LC1) concentration of 500 mg/ l , which corresponded to 1.73- and 1.83-fold increases over that produced with the media at Gal : Glu ratios of 1 : 9 and 9 : 1, respectively.

Human αs1-casein was expressed efficiently in Escherichia coli. The overproduced recombinant human a αs1-casein was about 25% of the total cell protein. Two different vectors were constructed to express Met-αs1-casein and Met-αs1-casein with a His-affinity tag at the C-terminus. Recombinant Met-s1-casein with a His-affinity tag was purified to homogeneity using Ni-nitrilotriacetic acid resin. N-terminal sequence of the first 10 amino acid residues of this purified protein was identical to that of mature human αs1-casein with an extra methionine residue at the N-terminus.

We developed the dual-micropillar-based microfluidic platform to direct embryonic stem (ES) cell fate. 4 × 4 dual-micropillar-based microfluidic platform consisted of 16 circular-shaped outer micropillars and 8 saddle-shaped inner micropillars in which single ES cells were cultured. We hypothesized that dual-micropillar arrays would play an important role in controlling the shear stress and cell docking. Circular-shaped outer micropillars minimized the shear stress, whereas saddle-shaped innermicropillars allowed for docking of individual ES cells. We observed the effect of saddle-shaped inner micropillars on cell docking in response to hydrodynamic resistance. We also demonstrated that ES cells cultured for 6 days within the dual-micropillar-based microfluidic platform differentiated into neural-like cells. Therefore, this dual-micropillar-based microfluidic platform could be a potentially powerful method for screening of lineage commitments of single ES cells.

We synthesized functional retinoic acid (RA)-polyethyleneimine (PEI) complex nanoparticles. NH groups of branched PEI chains were electrostatically interacted with carboxyl groups of RA surfaces to form cationic RA-PEI complex nanoparticles. We observed that the average diameter of RA-PEI complex nanoparticles was approximately 70 nm and the morphology of complex nanoparticles was homogeneous circular shape. To confirm the synthesis of RA-PEI complex nanoparticles, we characterized complex nanoparticles using (1)H nuclear magnetic resonance (NMR), indicating that hydrophilic branched PEI chains were covered on hydrophobic RA surfaces. Furthermore, we demonstrated that pH enabled the control of amounts of RA released from RA-PEI complex nanoparticles, showing that RA exposed to acidic pH 5 was steadily released (∼76%) from complex nanoparticles, whereas RA was rapidly released (∼97%) at pH 7.4 on day 11. We also observed that RA-PEI complex nanoparticles induced embryonic stem (ES) cell-derived neuronal differentiation. Therefore, this RA-PEI complex nanoparticle is a potentially powerful tool for directing murine ES cell fate.

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment.

We developed a new detection method for L-Dopa decarboxylase (DDC) activity using gold nanoparticles (AuNPs). When L-3,4-dihydroxyphenylalanine (L-Dopa) is decarboxylated to dopamine (DA) by DDC, DA induces the aggregation of AuNPs, and the color of the AuNPs changes from red to blue.

In this paper, we demonstrate a simple technique for sequentially introducing multiple sample liquids into microchannels driven by centrifugal force combined with a hydrophobic barrier pressure and apply the technique for performing solid-phase based on-chip DNA purification. Three microchannels with varying widths, all equipped with independent sample reservoirs at the inlets, were fabricated on a hydrophobic elastomer, poly(dimethylsiloxane) (PDMS). First, glass beads were packed inside the reaction chamber, and a whole cell containing the DNA extract was introduced into the widest channel by applying centrifugal force for physical adsorption of the DNA onto the glass beads. Next, washing and elution solutions were sequentially introduced into the intermediate and narrowest microchannels, respectively, by gradually increasing the amount of centrifugal force. Through a precise manipulation of the centrifugal force, the DNA adsorbed onto the glass beads was successfully washed and eluted in a continuous manner without the need to introduce each solution manually. A stepwise injection of liquids was successfully demonstrated using multiple ink solutions, the results of which corresponded well with the theoretical analyses. As a practical application, the D1S80 locus of human genomic DNA, which is widely used for forensic purposes, was successfully purified using the microdevice introduced in this study, as demonstrated through successful target amplification. This will pave the way for the construction of a control-free valve system for realizing on-chip DNA purification, which is one of the most labor-intensive and hard-to-miniaturize components, on a greatly simplified and miniaturized platform employing hydrophobic PDMS.

Cell penetrating peptides (CPPs) have been used to transport macromolecules into cells. Most CPPs have properties such as a strong polycationic charge, amphipathic basic, and hydrophobicity. In this study, we designed the peptides with multiple motifs composed of RGD and its analogs to induce integrin-mediated endocytosis as well as endosomal escape by forming an amphipathic helix in acidic endosomes. These peptides were proved less toxic to animal cells than those without acidic residues. Unexpectedly, peptide conjugated liposomes could penetrate into cells regardless of integrins. The replacement of all aspartic acids by glutamic acids did not prevent the peptide-mediated liposome uptake, and the higher basic and leucine contents enhanced the gene silencing activity of siRNA encapsulated in the liposomes. The peptide is considered to be a new type of CPP which can be used for drug delivery.

Cell-based immunotherapy has emerged as a promising therapy for the treatment of cancer. Measuring the changes in tumor volume and tumor markers post treatment has been the most common means of evaluating the therapeutic efficacy. In order to assess the consequences of a given therapy in real time, the development of efficient molecular probes and imaging modalities are urgently needed. Efficient molecular probes and imaging modalities will provide qualitative and quantitative real-time images with long-term stability in physiological conditions as well as low toxicity and high sensitivity for in vivo monitoring of the transplanted cells. Therapeutic cells can be intrinsically or extrinsically modified with proper molecular probes, amplified in vitro, and transferred back into the host. In this chapter, we will discuss the relative strengths and weaknesses of multiple molecular imaging modalities as well as recent advances in molecular imaging probes. We will also address their application in relation to in vivo tracking of dendritic cells (DCs), natural killer (NK) cells, and T cells. Noninvasive molecular imaging techniques have great potential in the diagnostic and prognostic assessments of patients.