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Biomedical subjects

Carl A Batt

Publications and source records attributed to Carl A Batt.

At least 19 recordsLinked to original sources
PubMed2006

Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris.

In Pichia pastoris, secretion of the A33 single-chain antibody fragment (A33scFv) was shown to reach levels of approximately 4 g l(-1) in fermentor cultures. In this study, we investigated whether manipulating chaperone and foldase levels in P. pastoris could further increase secretion of A33scFv. Cells were engineered to cooverexpress immunoglobulin binding protein (BiP) and/or protein disulfide isomerase (PDI) with A33scFv during growth in methanol as the sole carbon and energy source. Cooverexpression of BiP resulted in increased secretion levels of A33scFv by approximately threefold. In contrast, cooverexpression of PDI had no apparent effect on secretion of A33scFv. In cells cooverexpressing BiP and PDI, A33scFv secretion did not increase and protein levels remained the same as the control strain. We believe that secretion of A33scFv is increased by cooverexpression of BiP as a result of an increase in folding capacity inside the endoplasmic reticulum (ER). In addition, lack of increased single-chain secretion when PDI is coexpressed was unexpected due to the presence of disulfide bonds in A33scFv. We also show that during PDI cooverexpression with the single-chain there is a sixfold increase in BiP levels, indicating that the former is possibly inducing an unfolded protein response due to excess chaperone and recombinant protein in the ER.

Amino Acid Sequence↗
PubMed2006

Optimized linkage and quenching strategies for quantum dot molecular beacons.

Quantum dot (QD) molecular beacons were explored for sequence-specific DNA detection. The effectiveness of multiple linkage strategies and fluorescence quenchers were compared in hybridization-based assays. To compare linkage strategies, covalent amide linkage and streptavidin-biotin binding were used to link semiconductor QDs to molecular beacon DNA. Amide-linked beacons showed a 57% greater fluorescence increase than streptavidin-linked beacons when hybridized to 200 pmol of target DNA. The specificity of the molecular beacons, however, was similar for both linkage methods. Hybridization of both QD molecular beacons with non-complementary target DNA resulted in approximately 50% lower fluorescence intensity than hybridization with complementary DNA. The effectiveness of different quencher moieties was also evaluated. Iowa Black and 1.4 nm Nanogold-quenched molecular beacons exhibited approximately 2-fold greater fluorescence increases than dabcyl-quenched beacons when hybridized to complementary target. Specificity for target DNA was also confirmed through hybridization assays with non-complementary DNA. To provide insight into differences between the QD molecular beacons and the linkage strategies used, the hydrodynamic radius of each was measured. These measurements indicated that the larger radius of the streptavidin QDs (13.5 nm) than the carboxyl QDs (7 nm) could have a negative effect on FRET-based quenching for QD molecular beacons. These data outline the importance of choosing proper linkage methods and quencher moieties for creating high-quality QD molecular beacons.

Base Sequence↗
PubMed2006

Bionanofabrication of metallic and semiconductor nanoparticle arrays using S-layer protein lattices with different lateral spacings and geometries.

Two-dimensional (2-D) surface layer (S-layer) protein lattices isolated from the gram-positive bacterium Deinococcus radiodurans and the acidothermophilic archaeon Sulfolobus acidocaldarius were investigated and compared for their ability to biotemplate the formation of self-assembled, ordered arrays of inorganic nanoparticles (NPs). The NPs employed for these studies included citrate-capped gold NPs and various species of CdSe/ZnS core/shell quantum dots (QDs). The QD nanocrystals were functionalized with different types of thiol ligands (negative- or positive-charged/short- or long-chain length) in order to render them hydrophilic and thus water-soluble. Transmission electron microscopy, Fourier transform analyses, and pair correlation function calculations revealed that ordered nanostructured arrays with a range of spacings (approximately 7-22 nm) and different geometrical arrangements could be fabricated through the use of the two types of S-layers. These results demonstrate that it is possible to exploit the physicochemical/structural diversity of prokaryotic S-layer scaffolds to vary the morphological patterning of nanoscale metallic and semiconductor NP arrays.

Cadmium Compounds↗
PubMed2006

Real-time analysis of enzymatic surface-initiated polymerization using surface plasmon resonance (SPR).

The kinetics of enzymatic surface-initiated polymerization of PHB on gold surface has been examined by SPR and the resultant polymer layers characterized by AFM and FT-IR spectrometry. The immobilized enzyme catalyzed surface-initiated polymerization of 3HB-CoA, resulting in the formation of a polymer brush on the surface. The rate of polymer growth from the surface was monitored by SPR in real-time. Polymer growth as measured by the increase in the resonance angle showed no apparent lag phase during the polymerization reaction. SPR analysis also revealed that the thickness of the polymer film could be controlled by varying the initial enzyme density on the surface. The average thicknesses of the PHB film after polymerization reaction were 95, 45 and 15 nm for the surfaces that were treated with 0.5, 0.3 and 0.1*10(-6) M of enzyme, respectively. The binding of PHA synthase at different concentration to the mixed SAMs and subsequent polymerization.

Acyltransferases↗
PubMed2006

Self-assembly of dendrimer-encapsulated nanoparticle arrays using 2-D microbial S-layer protein biotemplates.

We investigated the formation of self-assembled two-dimensional (2-D) arrays of dendrimer-encapsulated platinum nanoparticles (Pt-DENs) using prokaryotic surface-layer (S-layer) proteins as biomacromolecular templates. The Pt-DENs (mean core diameter 1.8 +/- 0.5 nm) were synthesized by chemical reduction of metal ion species complexed within the interior of fourth-generation, hydroxyl-terminated, starburst poly(amidoamine) dendrimers (G4 PAMAM-OH). Detailed structural and elemental composition analyses performed using high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy, electron energy loss spectroscopy, and X-ray photoelectron spectroscopy indicated that the dendrimer-metal nanocomposite particles were crystalline in nature rather than amorphous and that at least some quantity of the platinum found within the particles is present in the expected zerovalent state. By using the S-layer lattices from the acidothermophilic archaeon Sulfolobus acidocaldarius and the Gram-positive bacterium Deinococcus radiodurans as a biotemplate, hexagonal- and honeycomb-ordered arrays of the Pt-DENs were successfully fabricated under a range of different pH conditions via noncovalent nanoparticle-protein interactions. Fast Fourier transform analyses of transmission electron microscopy images verified that the fabricated Pt-DEN assemblies displayed mean periodicities that corresponded well with the lattice constants of the native protein templates (i.e., 22 and 18 nm for S. acidocaldarius and D. radiodurans S layers, respectively). Our results demonstrate that utilizing pre-synthesized Pt-DENs in conjunction with microbial S-layer proteins displaying highly periodic topochemical properties can be an effective, novel route for creating patterned arrays of Pt nanoparticles with potential technological applications.

Adsorption↗
PubMed2005

Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha.

Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (P(phb)), acetoin (P(acoE), P(acoD), and P(acoX)) or pyruvate (P(pdhE)) metabolism were constructed and tested. The promoters were representative either of the enterobacterial sigma70 (P(phb), P(acoE), and P(pdhE))- or sigma54 (P(acoD) and P(acoX))-dependent promoters. To obtain polymers consisting of C4-C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 (Ps)) tandemly linked to the W. eutropha genes encoding beta-ketothiolase (phbA (We)) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB (We)) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the P(acoD) and P(acoX) promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters-independent of the addition of acetoin-resulted in the production of PHA polymers with high 3HB fractions (90-100 mol%) and with high 3HO contents (70-86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the P(acoE) promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.

Acetoin↗
PubMed2005

Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2.

A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha ( Ps-1) and pha ( Ps-2)) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha ( Ps-2) locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1( Ps-2)-phaC1 ( Ps-2)-phaZ ( Ps-2)-phaC2 ( Ps-2)-phaD ( Ps-2). The pha ( Ps-1) locus that contains phaC1 ( Ps-1)-phaZ ( Ps-1) appears to have arisen by a duplication event that placed it downstream of a gene (ORF1( Ps-1)), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 ( Ps-1) and phaC1 ( Ps-2) were investigated by heterologous expression in Wautersia eutropha PHB(-)4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1( Ps-1) to confer PHA synthesis, however, appeared reduced compared to that of PhaC1( Ps-2), since cells harboring PhaC1( Ps-1) accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1( Ps-2). Primary sequence analysis revealed that PhaC1( Ps-1) had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1( Ps-2)). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 ( Ps-1) produces a protein product in the native strain, it is likely that PhaC1( Ps-1) may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.

Acyltransferases↗
PubMed2004

Intercalation, delivery, and expression of the gene encoding green fluorescence protein utilizing nanobiohybrids.

Non-viral gene vectors have attracted much attention recently due to setbacks with viral delivery systems. Here a new non-viral delivery system based on nanobiohybrids synthesized by the intercalation of a full gene and promoter encoding Green Fluorescent Protein (GFP) between the layers of an inorganic host is reported. The nanobiohybrids were delivered to 9L glioma cells, JEG3 choriocarcinoma placental cells, and cardiac myocytes. All cells were able to internalize and tolerate the nanobiohybrids. In addition, all cells expressed the gene with some cell lines having up to 90% transfection efficiency. This new, bio-mimetic delivery system shows promise for use in non-viral gene therapy.

Animals↗
PubMed2004

Real-Time, label-free monitoring of tumor antigen and serum antibody interactions.

Conventional techniques for the detection of biomolecular interactions can be limited by the need for exogenous labels, time- and labor-intensive protocols, as well as by poor sensitivity levels. A refractometer instrument has been reconfigured to detect biomolecular interactions through changes in surface plasmon resonance (SPR). The binding kinetics and affinity values of anti-NY-ESO-1 monoclonal antibody, ES121, to the cancer-testis antigen NY-ESO-1 were determined according to the surface heterogeneity model and resulted in K(D) values of 1.3x10(-9) and 2.1x10(-10) M. The reconfigured instrument was then used to measure the interaction between tumor antigens and serum antibodies against these antigens in preselected cancer patient sera samples. The tumor antigens assayed included NY-ESO-1, SSX2 and p53, all used as recombinant proteins containing polyhistidine tags. These results demonstrated that the instrument is capable of detecting the binding of serum antibodies from cancer patient sera to immobilized tumor antigens, consistent with those observed previously in ELISA-based experiments. These results demonstrate the potential of SPR technology for the rapid diagnosis and monitoring immune responses.

Antibodies, Monoclonal↗
PubMed2004

Dendrimer-functionalized self-assembled monolayers as a surface plasmon resonance sensor surface.

We report here a multistep route for the immobilization of DNA and proteins on chemically modified gold substrates using fourth-generation NH(2)-terminated poly(amidoamine) dendrimers supported by an underlying amino undecanethiol (AUT) self-assembled monolayer (SAM). Bioactive ultrathin organic films were prepared via layer-by-layer self-assembly methods and characterized by fluorescence microscopy, variable angle spectroscopic ellipsometry, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR). The thickness of the AUT SAM base layer on the gold substrates was determined to be 1.3 nm from ellipsometry. Fluorescence microscopy and AFM measurements, in combination with analyses of the XPS/ATR-FTIR spectra, confirmed the presence of the dendrimer/biopolymer molecules on the multilayer sensor surfaces. Model proteins, including streptavidin and rabbit immunoglobulin proteins, were covalently attached to the dendrimer layer using linear cross-linking reagents. Through surface plasmon resonance measurements, we found that sensor surfaces containing a dendrimer layer displayed an increased protein immobilization capacity, compared to AUT SAM sensor surfaces without dendrimer molecules. Other SPR studies also revealed that the dendrimer-based surfaces are useful for the sensitive and specific detection of DNA-DNA interactions. Significantly, the multicomponent films displayed a high level of stability during repeated regeneration and hybridization cycles, and the kinetics of the DNA-DNA hybridization process did not appear to be influenced by surface mass transport limiting effects.

Biosensing Techniques↗
PubMed2004

An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris.

The expression of a humanized single-chain variable domain fragment antibody (A33scFv) was optimized for Pichia pastoris with yields exceeding 4 g L(-1). A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for immunotherapy of colon cancer. P. pastoris with a MutS phenotype was selected to express A33scFv, which was cloned under regulation of the methanol-inducible AOX1 promoter. We report the optimization of A33scFv production by examining methanol concentrations using fermentation technology with an on-line methanol control in fed-batch fermentation of P. pastoris. In addition, we examined the effect of pH on A33scFv production and biomass accumulation during the methanol induction phase. A33scFv production was found to increase with higher methanol concentrations, reaching 4.3 g L(-1) after 72 h induction with 0.5% (v/v) methanol. Protein production was also greatly affected by pH, resulting in higher yields (e.g., 4.88 g L(-1)) at lower pH values. Biomass accumulation did not seem to vary when cells were induced at different pH values, but was greatly affected by lower concentration of methanol. Purification of A33scFv from clarified medium was done using a two-step chromatographic procedure using anion-exchange and hydrophobic interaction chromatography, resulting in 25% recovery and >90% purity. Pure A33scFv was tested for functionality using surface plasmon resonance and showed activity against immobilized A33 antigen. Our results demonstrate that functional A33scFv can be produced in sufficient quantities using P. pastoris for use in further functionality studies and diagnostic applications.

Animals↗
PubMed2004

Engineering of chimeric class II polyhydroxyalkanoate synthases.

PHA synthase is a key enzyme involved in the biosynthesis of polyhydroxyalkanoates (PHAs). Using a combinatorial genetic strategy to create unique chimeric class II PHA synthases, we have obtained a number of novel chimeras which display improved catalytic properties. To engineer the chimeric PHA synthases, we constructed a synthetic phaC gene from Pseudomonas oleovorans (phaC1Po) that was devoid of an internal 540-bp fragment. Randomly amplified PCR products (created with primers based on conserved phaC sequences flanking the deleted internal fragment) were generated using genomic DNA isolated from soil and were substituted for the 540-bp internal region. The chimeric genes were expressed in a PHA-negative strain of Ralstonia eutropha, PHB(-)4 (DSM 541). Out of 1,478 recombinant clones screened for PHA production, we obtained five different chimeric phaC1Po genes that produced more PHA than the native phaC1Po. Chimeras S1-71, S4-8, S5-58, S3-69, and S3-44 exhibited 1.3-, 1.4-, 2.0-, 2.1-, and 3.0-fold-increased levels of in vivo activity, respectively. All of the mutants mediated the synthesis of PHAs with a slightly increased molar fraction of 3-hydroxyoctanoate; however, the weight-average molecular weights (Mw) of the PHAs in all cases remained almost the same. Based upon DNA sequence analyses, the various phaC fragments appear to have originated from Pseudomonas fluorescens and Pseudomonas aureofaciens. The amino acid sequence analyses showed that the chimeric proteins had 17 to 20 amino acid differences from the wild-type phaC1Po, and these differences were clustered in the same positions in the five chimeric clones. A threading model of PhaC1Po, developed based on homology of the enzyme to the Burkholderia glumae lipase, suggested that the amino acid substitutions found in the active chimeras were located mostly on the protein model surface. Thus, our combinatorial genetic engineering strategy proved to be broadly useful for improving the catalytic activities of PHA synthase enzymes.

Acyltransferases↗
PubMed2004

Restoration of a defective Lactococcus lactis xylose isomerase.

The genes (xylA) encoding xylose isomerase (XI) from two Lactococcus lactis subsp. lactis strains, 210 (Xyl(-)) and IO-1 (Xyl(+)), were cloned, and the activities of their expressed proteins in recombinant strains of Escherichia coli were investigated. The nucleotide and amino acid sequence homologies between the xylA genes were 98.4 and 98.6%, respectively, and only six amino acid residues differed between the two XIs. The purified IO-1 XI was soluble with K(m) and k(cat) being 2.25 mM and 184/s, respectively, while the 210 XI was insoluble and inactive. Site-directed mutagenesis on 210 xylA showed that a triple mutant possessing R202M/Y218D/V275A mutations regained XI activity and was soluble. The K(m) and k(cat) of this mutant were 4.15 mM and 141/s, respectively. One of the IO-1 XI mutants, S388T, was insoluble and showed negligible activity similar to that of 210 XI. The introduction of a K407E mutation to the IO-1 S388T XI mutant restored its activity and solubility. The dissolution of XI activity in L. lactis subsp. lactis involves a series of mutations that collectively eliminate enzyme activity by reducing the solubility of the enzyme.

Aldose-Ketose Isomerases↗
PubMed2003

Nucleic acid purification using microfabricated silicon structures.

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.

Adsorption↗
PubMed2003

Reversible unfolding of bovine beta-lactoglobulin mutants without a free thiol group.

Bovine beta-lactoglobulin (beta-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three beta-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type beta-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type beta-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type beta-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of beta-lg more exactly under various conditions.

Amino Acid Substitution↗