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1.  Single-chain Fv phage display propensity exhibits strong positive correlation with overall expression levels 
BMC Biotechnology  2008;8:97.
Single chain Fvs (scFvs) are widely applied in research, diagnostics and therapeutic settings. Display and selection from combinatorial libraries is the main route to their discovery and many factors influence the success of this process. They exhibit low thermodynamic stability, resulting in low levels of premature cytosolic folding or aggregation which facilitates sec YEG-mediated translocation and phage in E. coli. However, there is little data analysing how this is related to and influenced by scFv protein expression.
We characterised the relationship between overall scFv expression and display propensity for a panel of 15 anti-tetanus toxin scFvs and found a strong positive correlation (Rho = 0.88, p < 0.005) between the two parameters. Display propensity, overall expression and soluble localisation to the periplasm and extracellular fractions were clone specific characteristics which varied despite high levels of sequence homology. There was no correlation between display of scFv or its expression in non-fused (free) form with soluble scFv localisation to the periplasm or culture supernatant. This suggests that divergence in the fate of scFv-pIII and non-fused scFv after translocation to the periplasm accounts for the observed disparity. Differential degrees of periplasmic aggregation of non-fused scFv between clones may affect the partitioning of scFv in the periplasm and culture supernatant abrogating any correlation. We suggest that these factors do not apply to the scFv-pIII fusion since it remains anchored to the bacterial inner membrane as part of the innate phage packaging and budding process.
We conclude that in the absence of premature cytosolic aggregation or folding, the propensity of a scFv to be displayed on phage is directly related to its overall expression level and is thus indirectly influenced by factors such as codon bias, mRNA abundance or putative DNA motifs affecting expression. This suggests that scFvs capable of high overall expression and display levels may not produce high yields of non phage-fused soluble protein in either the periplasmic or extracellular fractions of E. coli. This should be considered when screening clones selected from combinatorial libraries for further study.
The nucleotide and amino acid sequences of the anti-tetanus toxin scFvs have been deposited in the EMBL data base: accession numbers-C1: AM749134, C2: AM749135, C3: AM749136, C4: AM749137, C5: AM749138, N1: AM749139, N2: AM749140, N3: AM749141, N4: AM749142, N5: AM749143 J1; AM749144, J2: AM749145, J3: AM749146, J4: AM749147, J5: AM749148.
PMCID: PMC2630973  PMID: 19113995
2.  A modeling study by response surface methodology and artificial neural network on culture parameters optimization for thermostable lipase production from a newly isolated thermophilic Geobacillus sp. strain ARM 
BMC Biotechnology  2008;8:96.
Thermostable bacterial lipases occupy a place of prominence among biocatalysts owing to their novel, multifold applications and resistance to high temperature and other operational conditions. The capability of lipases to catalyze a variety of novel reactions in both aqueous and nonaqueous media presents a fascinating field for research, creating interest to isolate novel lipase producers and optimize lipase production. The most important stages in a biological process are modeling and optimization to improve a system and increase the efficiency of the process without increasing the cost.
Different production media were tested for lipase production by a newly isolated thermophilic Geobacillus sp. strain ARM (DSM 21496 = NCIMB 41583). The maximum production was obtained in the presence of peptone and yeast extract as organic nitrogen sources, olive oil as carbon source and lipase production inducer, sodium and calcium as metal ions, and gum arabic as emulsifier and lipase production inducer. The best models for optimization of culture parameters were achieved by multilayer full feedforward incremental back propagation network and modified response surface model using backward elimination, where the optimum condition was: growth temperature (52.3°C), medium volume (50 ml), inoculum size (1%), agitation rate (static condition), incubation period (24 h) and initial pH (5.8). The experimental lipase activity was 0.47 Uml-1 at optimum condition (4.7-fold increase), which compared well to the maximum predicted values by ANN (0.47 Uml-1) and RSM (0.476 Uml-1), whereas R2 and AAD were determined as 0.989 and 0.059% for ANN, and 0.95 and 0.078% for RSM respectively.
Lipase production is the result of a synergistic combination of effective parameters interactions. These parameters are in equilibrium and the change of one parameter can be compensated by changes of other parameters to give the same results. Though both RSM and ANN models provided good quality predictions in this study, yet the ANN showed a clear superiority over RSM for both data fitting and estimation capabilities. On the other hand, ANN has the disadvantage of requiring large amounts of training data in comparison with RSM. This problem was solved by using statistical experimental design, to reduce the number of experiments.
PMCID: PMC2637859  PMID: 19105837
3.  Hairy-root organ cultures for the production of human acetylcholinesterase 
BMC Biotechnology  2008;8:95.
Human cholinesterases can be used as a bioscavenger of organophosphate toxins used as pesticides and chemical warfare nerve agents. The practicality of this approach depends on the availability of the human enzymes, but because of inherent supply and regulatory constraints, a suitable production system is yet to be identified.
As a promising alternative, we report the creation of "hairy root" organ cultures derived via Agrobacterium rhizogenes-mediated transformation from human acetylcholinesterase-expressing transgenic Nicotiana benthamiana plants. Acetylcholinesterase-expressing hairy root cultures had a slower growth rate, reached to the stationary phase faster and grew to lower maximal densities as compared to wild type control cultures. Acetylcholinesterase accumulated to levels of up to 3.3% of total soluble protein, ~3 fold higher than the expression level observed in the parental plant. The enzyme was purified to electrophoretic homogeneity. Enzymatic properties were nearly identical to those of the transgenic plant-derived enzyme as well as to those of mammalian cell culture derived enzyme. Pharmacokinetic properties of the hairy-root culture derived enzyme demonstrated a biphasic clearing profile. We demonstrate that master banking of plant material is possible by storage at 4°C for up to 5 months.
Our results support the feasibility of using plant organ cultures as a successful alternative to traditional transgenic plant and mammalian cell culture technologies.
PMCID: PMC2648960  PMID: 19105816
4.  Better estimation of protein-DNA interaction parameters improve prediction of functional sites 
BMC Biotechnology  2008;8:94.
Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method designed to produce many such sequences.
We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP), and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones) in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay) and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation) trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites.
We think that training binding site detection algorithms on datasets from binding assays lead to better prediction. The improvements in accuracy came from the unbiased nature of the SELEX dataset rather than from the number of sites available. We believe that with progress in short-read sequencing technology, one could use SELEX methods to characterize binding affinities of many low specificity transcription factors.
PMCID: PMC2654563  PMID: 19105805
5.  Simultaneous tracking of fly movement and gene expression using GFP 
BMC Biotechnology  2008;8:93.
Green Fluorescent Protein (GFP) is used extensively as a reporter for transgene expression in Drosophila and other organisms. However, GFP has not generally been used as a reporter for circadian patterns of gene expression, and it has not previously been possible to correlate patterns of reporter expression with 3D movement and behavior of transgenic animals.
We present a video tracking system that allows tissue-specific GFP expression to be quantified and correlated with 3D animal movement in real time. eyeless/Pax6 reporter expression had a 12 hr period that correlated with fly activity levels.
hsp70 and hsp22 gene reporters were induced during fly aging in circadian patterns (24 hr and 18 hr periods, respectively), and spiked in the hours preceding and overlapping the death of the animal. The phase of hsp gene reporter expression relative to fly activity levels was different for each fly, and remained the same throughout the life span.
These experiments demonstrate that GFP can readily be used to assay longitudinally fly movement and tissue-specific patterns of gene expression. The hsp22-GFP and hsp70-GFP expression patterns were found to reflect accurately the endogenous gene expression patterns, including induction during aging and circadian periodicity. The combination of these new tracking methods with the hsp-GFP reporters revealed additional information, including a spike in hsp22 and hsp70 reporter expression preceding death, and an intriguing fly-to-fly variability in the phase of hsp70 and hsp22 reporter expression patterns. These methods allow specific temporal patterns of gene expression to be correlated with temporal patterns of animal activity, behavior and mortality.
PMCID: PMC2625341  PMID: 19087237
6.  Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors 
BMC Biotechnology  2008;8:92.
Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in E. coli have been unsuccessful.
Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in E. coli and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of E. coli culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (kcat/KM) of the precursor proteins by two orders of magnitude.
A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in E. coli. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.
PMCID: PMC2621203  PMID: 19077216
7.  An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol 
BMC Biotechnology  2008;8:91.
Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.
We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.
Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™) but increased the overall success rates.
PMCID: PMC2629768  PMID: 19055817
8.  Reliable microRNA profiling in routinely processed formalin-fixed paraffin-embedded breast cancer specimens using fluorescence labelled bead technology 
BMC Biotechnology  2008;8:90.
During the last years the analysis of microRNA expression patterns has led to completely new insights into cancer biology. Furthermore, these patterns are a very promising tool for the development of new diagnostic and prognostic markers. However, most human tumour samples for which long term clinical records are available exist only as formalin-fixed paraffin-embedded specimens. Therefore, the aim of this study was to examine the feasibility of microRNA profiling studies in routinely processed formalin-fixed paraffin-embedded human breast cancer specimens using fluorescence labelled bead technology.
A statistically highly significant correlation (Spearman r: 0.78 – 0.90, p < 0.0001) was observed for the expression of 319 microRNAs in routinely processed FFPE breast cancer specimens and paired fresh frozen tissue samples (n = 5). Results were confirmed in a larger series analyzing a selection of 10 microRNAs reported to be deregulated in breast cancer (n = 12). The expression pattern of 3 microRNAs was independently validated in this cohort using real-time RT-PCR technology.
Comprehensive microRNA expression patterns can be reliably derived from routinely processed FFPE breast cancer specimens using fluorescence labelled bead technology.
PMCID: PMC2605753  PMID: 19038028
9.  Preparation of alginate coated chitosan microparticles for vaccine delivery 
BMC Biotechnology  2008;8:89.
Absorption of antigens onto chitosan microparticles via electrostatic interaction is a common and relatively mild process suitable for mucosal vaccine. In order to increase the stability of antigens and prevent an immediate desorption of antigens from chitosan carriers in gastrointestinal tract, coating onto BSA loaded chitosan microparticles with sodium alginate was performed by layer-by-layer technology to meet the requirement of mucosal vaccine.
The prepared alginate coated BSA loaded chitosan microparticles had loading efficiency (LE) of 60% and loading capacity (LC) of 6% with mean diameter of about 1 μm. When the weight ratio of alginate/chitosan microparticles was greater than 2, the stable system could be obtained. The rapid charge inversion of BSA loaded chitosan microparticles (from +27 mv to -27.8 mv) was observed during the coating procedure which indicated the presence of alginate layer on the chitosan microparticles surfaces. According to the results obtained by scanning electron microscopy (SEM), the core-shell structure of BSA loaded chitosan microparticles was observed. Meanwhile, in vitro release study indicated that the initial burst release of BSA from alginate coated chitosan microparticles was lower than that observed from uncoated chitosan microparticles (40% in 8 h vs. about 84% in 0.5 h). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) assay showed that alginate coating onto chitosan microparticles could effectively protect the BSA from degradation or hydrolysis in acidic condition for at least 2 h. The structural integrity of alginate modified chitosan microparticles incubated in PBS for 24 h was investigated by FTIR.
The prepared alginate coated chitosan microparticles, with mean diameter of about 1 μm, was suitable for oral mucosal vaccine. Moreover, alginate coating onto the surface of chitosan microparticles could modulate the release behavior of BSA from alginate coated chitosan microparticles and could effectively protect model protein (BSA) from degradation in acidic medium in vitro for at least 2 h. In all, the prepared alginate coated chitosan microparticles might be an effective vehicle for oral administration of antigens.
PMCID: PMC2603011  PMID: 19019229
10.  A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) 
BMC Biotechnology  2008;8:87.
Human glutamic acid decarboxylase 65 (hGAD65) is a key autoantigen in type 1 diabetes, having much potential as an important marker for the prediction and diagnosis of type 1 diabetes, and for the development of novel antigen-specific therapies for the treatment of type 1 diabetes. However, recombinant production of hGAD65 using conventional bacterial or mammalian cell culture-based expression systems or nuclear transformed plants is limited by low yield and low efficiency. Chloroplast transformation of the unicellular eukaryotic alga Chlamydomonas reinhardtii may offer a potential solution.
A DNA cassette encoding full-length hGAD65, under the control of the C. reinhardtii chloroplast rbcL promoter and 5'- and 3'-UTRs, was constructed and introduced into the chloroplast genome of C. reinhardtii by particle bombardment. Integration of hGAD65 DNA into the algal chloroplast genome was confirmed by PCR. Transcriptional expression of hGAD65 was demonstrated by RT-PCR. Immunoblotting verified the expression and accumulation of the recombinant protein. The antigenicity of algal-derived hGAD65 was demonstrated with its immunoreactivity to diabetic sera by ELISA and by its ability to induce proliferation of spleen cells from NOD mice. Recombinant hGAD65 accumulated in transgenic algae, accounts for approximately 0.25–0.3% of its total soluble protein.
Our results demonstrate the potential value of C. reinhardtii chloroplasts as a novel platform for rapid mass production of immunologically active hGAD65. This demonstration opens the future possibility for using algal chloroplasts as novel bioreactors for the production of many other biologically active mammalian therapeutic proteins.
PMCID: PMC2621204  PMID: 19014643
11.  Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems 
BMC Biotechnology  2008;8:88.
Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures.
To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression profile of a PAP85::GUS transgene highly corresponds with native PAP85 expression.
We describe a novel combination of the favourable attributes of the Gateway and recombineering systems to enable efficient cloning and manipulation of genomic DNA clones for more effective characterisation of gene function. Although the system and plasmid vectors described here were developed for applications in plants, the general approach is broadly applicable to gene characterisation studies in many biological systems.
PMCID: PMC2601046  PMID: 19014699
12.  Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions 
BMC Biotechnology  2008;8:86.
Array-based comparative genomic hybridization (CGH) and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5) are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb). Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3) ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays.
Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC) arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and data quality from gene expression experiments.
HyPer5 is a red fluorescent dye that behaves functionally similar to Cy5 except in stability to ozone and light. HyPer5 is demonstrated to be resistant to ozone at up to 300 ppb, levels significantly higher than commonly observed during summer months. Consequently, HyPer5 dye can be used in parallel with Cy3 under any environmental conditions in array experiments.
PMCID: PMC2613886  PMID: 19014508
13.  Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells 
BMC Biotechnology  2008;8:85.
Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.
Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients.
The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated.
The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.
PMCID: PMC2631500  PMID: 19014469
14.  Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications 
BMC Biotechnology  2008;8:84.
Commercially available curcumin preparations contain a mixture of related polyphenols, collectively referred to as curcuminoids. These encompass the primary component curcumin along with its co-purified derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids have numerous biological activities, including inhibition of cancer related cell proliferation and reduction of amyloid plaque formation associated with Alzheimer disease. Unfortunately, the solubility of curcuminoids in aqueous solutions is exceedingly low. This restricts their systemic availability in orally administered formulations and limits their therapeutic potential.
Methods are described that achieve high concentrations of soluble curcuminoids in serum. Solid curcuminoids were either mixed directly with serum, or they were predissolved in dimethyl sulfoxide and added as aliquots to serum. Both methods resulted in high levels of curcuminoid-solubility in mammalian sera from different species. However, adding aliquots of dimethyl sulfoxide-dissolved curcuminoids to serum proved to be more efficient, producing soluble curcuminoid concentrations of at least 3 mM in human serum. The methods also resulted in the differential solubility of individual curcuminoids in serum. The addition of dimethyl sulfoxide-dissolved curcuminoids to serum preferentially solubilized curcumin, whereas adding solid curcuminoids predominantly solubilized bisdemethoxycurcumin. Either method of solubilization was equally effective in inhibiting dose-dependent HeLa cell proliferation in culture. The maximum concentration of curcuminoids achieved in serum was at least 100-fold higher than that required for inhibiting cell proliferation in culture and 1000-fold higher than the concentration that has been reported to prevent amyloid plaque formation associated with Alzheimer disease. Curcuminoids were also highly soluble in solutions of purified albumin, a major component of serum.
These results suggest the possibility of alternative therapeutic approaches by injection or infusion of relatively small amounts of curcuminoid-enriched serum. They also provide tools to reproducibly solubilize curcuminoids for analysis in cell culture applications. The differential solubility of curcuminoids achieved by different methods of solubilization offers convenient alternatives to assess the diverse biological effects contributed by curcumin and its derivatives.
PMCID: PMC2612664  PMID: 18990234
15.  Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid 
BMC Biotechnology  2008;8:83.
Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.
Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.
The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.
PMCID: PMC2588579  PMID: 18983675
16.  Recombinant amyloid beta-peptide production by coexpression with an affibody ligand 
BMC Biotechnology  2008;8:82.
Oligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation.
Coexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40) and Aβ(1–42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Aβ(1–42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies.
The coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Aβ(1–42) is reported.
PMCID: PMC2606684  PMID: 18973685
17.  Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A 
BMC Biotechnology  2008;8:79.
Pseudomonas fluorescens is an important food spoilage organism, usually found in the form of biofilms. Bacterial biofilms are inherently resistant to a variety of antimicrobial agents, therefore alternative methods to biofilm control, such as bacteriophages (phages) have been suggested. Phage behavior on biofilms is still poorly investigated and needs further understanding. Here we describe the application of phage ϕIBB-PF7, a newly isolated phage, to control P. fluorescens biofilms. The biofilms were formed under static or dynamic conditions and with or without renewal of medium.
Conditions for biofilm formation influenced the feature of the biofilm and the morphology of P. fluorescens. Biomass removal due to phage activity varied between 63 and 91% depending on the biofilm age and the conditions under which the biofilm had been formed and phages applied. Removal of the biofilm by phage treatment was faster in younger biofilms, but the same number of surviving cells was detected in all tested biofilms, after only 4 h of treatment, even in older biofilms. Under static conditions, a 3 log higher number of phage progeny remained either inside the biofilm matrix or attached to the substratum surface than under dynamic conditions, pointing to the importance of experimental conditions for the efficacy of phage entrapment into the biofilm.
Phage ϕIBB-PF7A is highly efficient in removing P. fluorescens biofilms within a short time interval. The conditions of biofilm formation and applied during phage infection are critical for the efficacy of the sanitation process. The integration of phages into the biofilm matrix and their entrapment to the surface may be further beneficial factors when phage treatment is considered alone or in addition to chemical biocides in industrial environments where P. fluorescens causes serious spoilage.
PMCID: PMC2584026  PMID: 18954451
18.  Isolation and characterization of a T7-like lytic phage for Pseudomonas fluorescens 
BMC Biotechnology  2008;8:80.
Despite the proven relevance of Pseudomonas fluorescens as a spoilage microorganism in milk, fresh meats and refrigerated food products and the recognized potential of bacteriophages as sanitation agents, so far no phages specific for P. fluorescens isolates from dairy industry have been closely characterized in view of their lytic efficiency. Here we describe the isolation and characterization of a lytic phage capable to infect a variety of P. fluorescens strains isolated from Portuguese and United States dairy industries.
Several phages were isolated which showed a different host spectrum and efficiency of lysis. One of the phages, phage ϕIBB-PF7A, was studied in detail due to its efficient lysis of a wide spectrum of P. fluorescens strains and ribotypes. Phage ϕIBB-PF7A with a head diameter of about 63 nm and a tail size of about 13 × 8 nm belongs morphologically to the Podoviridae family and resembles a typical T7-like phage, as analyzed by transmission electron microscopy (TEM). The phage growth cycle with a detected latent period of 15 min, an eclipse period of 10 min, a burst size of 153 plaque forming units per infected cell, its genome size of approximately 42 kbp, and the size and N-terminal sequence of one of the protein bands, which gave similarity to the major capsid protein 10A, are consistent with this classification.
The isolated T7-like phage, phage ϕIBB-PF7A, is fast and efficient in lysing different P. fluorescens strains and may be a good candidate to be used as a sanitation agent to control the prevalence of spoilage causing P. fluorescens strains in dairy and food related environments.
PMCID: PMC2582237  PMID: 18954452
19.  Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos 
BMC Biotechnology  2008;8:81.
The dual-luciferase assay has been widely used in cell lines to determine rapidly but accurately the activity of a given promoter. Although this strategy has proved very useful, it does not allow the promoter and gene function to be analyzed in the context of the whole organism.
Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.
This assay has several advantages compared with the classical in vitro (cell lines) and in vivo (transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.
PMCID: PMC2596795  PMID: 18954456
20.  Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence 
BMC Biotechnology  2008;8:78.
Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an α-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface.
The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM.
The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.
PMCID: PMC2577101  PMID: 18854017
21.  A construct with fluorescent indicators for conditional expression of miRNA 
BMC Biotechnology  2008;8:77.
Transgenic RNAi holds promise as a simple, low-cost, and fast method for reverse genetics in mammals. It may be particularly useful for producing animal models for hypomorphic gene function. Inducible RNAi that permits spatially and temporally controllable gene silencing in vivo will enhance the power of transgenic RNAi approach. Furthermore, because microRNA (miRNA) targeting specific genes can be expressed simultaneously with protein coding genes, incorporation of fluorescent marker proteins can simplify the screening and analysis of transgenic RNAi animals.
We sought to optimally express a miRNA simultaneously with a fluorescent marker. We compared two construct designs. One expressed a red fluorescent protein (RFP) and a miRNA placed in its 3' untranslated region (UTR). The other expressed the same RFP and miRNA, but the precursor miRNA (pre-miRNA) coding sequence was placed in an intron that was inserted into the 3'-UTR. We found that the two constructs expressed comparable levels of miRNA. However, the intron-containing construct expressed a significantly higher level of RFP than the intron-less construct. Further experiments indicate that the 3'-UTR intron enhances RFP expression by its intrinsic gene-expression-enhancing activity and by eliminating the inhibitory effect of the pre-miRNA on the expression of RFP. Based on these findings, we incorporated the intron-embedded pre-miRNA design into a conditional expression construct that employed the Cre-loxP system. This construct initially expressed EGFP gene, which was flanked by loxP sites. After exposure to Cre recombinase, the transgene stopped EGFP expression and began expression of RFP and a miRNA, which silenced the expression of specific cellular genes.
We have designed and tested a conditional miRNA-expression construct and showed that this construct expresses both the marker genes strongly and can silence the target gene efficiently upon Cre-mediated induction of the miRNA expression. This construct can be used to increase the efficiency of making cell lines or transgenic animals that stably express miRNA targeting specific genes.
PMCID: PMC2569932  PMID: 18840295
22.  One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester 
BMC Biotechnology  2008;8:76.
Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.
A novel redox buffer consisting of MESNA and diMESNA showed a refolding efficiency comparable to that of GSH/GSSG and prevented loss of the protein's thioester functionality. Moreover, introduction of the MESNA/diMESNA redox couple in the cleavage buffer allowed simultaneous on-column refolding of Ribonuclease A and intein-mediated cleavage to yield Ribonuclease A with a C-terminal MESNA-thioester. The C-terminal thioester was shown to be active in native chemical ligation.
An efficient method was developed for the production of disulfide bond containing proteins with C-terminal thioesters. Introduction of a MESNA/diMESNA redox couple resulted in simultaneous on-column refolding, purification and thioester generation of the model protein Ribonuclease A.
PMCID: PMC2570673  PMID: 18828922
23.  Pharmaceutical induction of ApoE secretion by multipotent mesenchymal stromal cells (MSCs) 
BMC Biotechnology  2008;8:75.
Apolipoprotein E (ApoE) is a molecular scavenger in the blood and brain. Aberrant function of the molecule causes formation of protein and lipid deposits or "plaques" that characterize Alzheimer's disease (AD) and atherosclerosis. There are three human isoforms of ApoE designated ε2, ε3, and ε4. Each isoform differentially affects the structure and function of the protein and thus the development of disease. Homozygosity for ApoE ε4 is associated with atherosclerosis and Alzheimer's disease whereas ApoE ε2 and ε3 tend to be protective. Furthermore, the ε2 form may cause forms of hyperlipoproteinemia. Therefore, introduction of ApoE ε3 may be beneficial to patients that are susceptible to or suffering from these diseases. Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) are adult progenitor cells found in numerous tissues. They are easily expanded in culture and engraft into host tissues when administered appropriately. Furthermore, MSCs are immunosuppressive and have been reported to engraft as allogeneic transplants. In our previous study, mouse MSCs (mMSCs) were implanted into the brains of ApoE null mice, resulting in production of small amounts of ApoE in the brain and attenuation of cognitive deficits. Therefore human MSCs (hMSCs) are a promising vector for the administration of ApoE ε3 in humans.
Unlike mMSCs, hMSCs were found not to express ApoE in culture; therefore a molecular screen was performed for compounds that induce expression. PPARγ agonists, neural stem cell conditioned medium, osteo-inductive media, dexamethasone, and adipo-inductive media (AIM) were tested. Of the conditions tested, only AIM or dexamethasone induced sustained secretion of ApoE in MSCs and the duration of secretion was only limited by the length of time MSCs could be sustained in culture. Upon withdrawal of the inductive stimuli, the ApoE secretion persisted for a further 14 days.
The data demonstrated that pre-treatment and perhaps co-administration of MSCs homozygous for ApoE ε3 and dexamethasone may represent a novel therapy for severe instances of AD, atherosclerosis and other ApoE-related diseases.
PMCID: PMC2596794  PMID: 18823563
24.  Inhibition of cell growth and invasion by epidermal growth factor-targeted phagemid particles carrying siRNA against focal adhesion kinase in the presence of hydroxycamptothecin 
BMC Biotechnology  2008;8:74.
Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery.
We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion.
These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.
PMCID: PMC2562374  PMID: 18801171
25.  Shewanella oneidensis: a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii 
BMC Biotechnology  2008;8:73.
The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.
Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.
In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.
PMCID: PMC2553784  PMID: 18801156

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