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1.  Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha 
BMC Biotechnology  2012;12:96.
Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.
The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.
The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.
PMCID: PMC3539880  PMID: 23253823
Staphylokinase; Hansenula polymorpha; Recombinant protein; Fermentation; Scale-up; HTP
2.  A vaccine grade of yeast Saccharomyces cerevisiae expressing mammalian myostatin 
BMC Biotechnology  2012;12:97.
Yeast Saccharomyces cerevisiae is a widely-used system for protein expression. We previously showed that heat-killed whole recombinant yeast vaccine expressing mammalian myostatin can modulate myostatin function in mice, resulting in increase of body weight and muscle composition in these animals. Foreign DNA introduced into yeast cells can be lost soon unless cells are continuously cultured in selection media, which usually contain antibiotics. For cost and safety concerns, it is essential to optimize conditions to produce quality food and pharmaceutical products.
We developed a simple but effective method to engineer a yeast strain stably expressing mammalian myostatin. This method utilized high-copy-number integration of myostatin gene into the ribosomal DNA of Saccharomyces cerevisiae. In the final step, antibiotic selection marker was removed using the Cre-LoxP system to minimize any possible side-effects for animals. The resulting yeast strain can be maintained in rich culture media and stably express mammalian myostatin for two years. Oral administration of the recombinant yeast was able to induce immune response to myostatin and modulated the body weight of mice.
Establishment of such yeast strain is a step further toward transformation of yeast cells into edible vaccine to improve meat production in farm animals and treat human muscle-wasting diseases in the future.
PMCID: PMC3543327  PMID: 23253888
Saccharomyces cerevisiae; Vaccine; Myostatin; Chromosomal integration
3.  Use of anionic denaturing detergents to purify insoluble proteins after overexpression 
BMC Biotechnology  2012;12:95.
Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification.
Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC.
Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.
PMCID: PMC3536628  PMID: 23231964
Inclusion Bodies; Sodiumdodecylsulphate (SDS); N-lauroylsarcosine sodium salt (Sarkosyl); Immobilized Metal Ion Affinity Chromatography (IMAC)
4.  Guanylate-binding protein 1 expression from embryonal endothelial progenitor cells reduces blood vessel density and cellular apoptosis in an axially vascularised tissue-engineered construct 
BMC Biotechnology  2012;12:94.
Guanylate binding protein-1 (GBP-1) is a large GTPase which is actively secreted by endothelial cells. It is a marker and intracellular inhibitor of endothelial cell proliferation, migration, and invasion. We previously demonstrated that stable expression of GBP-1 in murine endothelial progenitor cells (EPC) induces their premature differentiation and decreases their migration capacity in vitro and in vivo. The goal of the present study was to assess the antiangiogenic capacity of EPC expressing GBP-1 (GBP-1-EPC) and their impact on blood vessel formation in an axially vascularized 3-D bioartificial construct in vivo.
Functional in vitro testing demonstrated a significant increase in VEGF secretion by GBP-1-EPC after induction of cell differentiation. Undifferentiated GBP-1-EPC, however, did not secrete increased levels of VEGF compared to undifferentiated control EPC expressing an empty vector (EV-EPC). In our In vivo experiments, we generated axially vascularized tissue-engineered 3-D constructs. The new vascular network arises from an arterio-venous loop (AVL) embedded in a fibrin matrix inside a separation chamber. Total surface area of the construct as calculated from cross sections was larger after transplantation of GBP-1-EPC compared to control EV-EPC. This indicated reduced formation of fibrovascular tissue and less resorption of fibrin matrix compared to constructs containing EV-EPC. Most notably, the ratio of blood vessel surface area over total construct surface area in construct cross sections was significantly reduced in the presence of GBP-1-EPC. This indicates a significant reduction of blood vessel density and thereby inhibition of blood vessel formation from the AVL constructs caused by GBP-1. In addition, GBP-1 expressed from EPC significantly reduced cell apoptosis compared to GBP-1-negative controls.
Transgenic EPC expressing the proinflammatory antiangiogenic GTPase GBP-1 can reduce blood vessel density and inhibit apoptosis in a developing bioartificial vascular network and may become a new powerful tool to manipulate angiogenetic processes in tissue engineering and other pathological conditions such as tumour angiogenesis.
PMCID: PMC3610105  PMID: 23217187
Angiogenesis; Endothelial progenitor cells; Guanylate-binding protein 1; In vivo tissue engineering
5.  Multimodal in vivo imaging reveals limited allograft survival, intrapulmonary cell trapping and minimal evidence for ischemia-directed BMSC homing 
BMC Biotechnology  2012;12:93.
Despite positive reports on the efficacy of stem cell therapy for the treatment of cardiovascular disease, the nature of stem cell homing to ischemic tissues remains elusive.
We used a mouse model of peripheral tissue ischemia to study the survival and homing capacity of dual reporter gene (eGFP/Luciferase) expressing bone marrow-derived stromal cells (BMSC). Cell homing and survival were studied in the presence and absence of ciclosporin A (CsA) immunosuppression using bioluminescence imaging (BLI) together with confocal endomicroscopy. Different injection strategies were applied: central venous (CV), intra-arterial (IA) and intramuscular (IM). BLI and confocal endomicroscopy evidenced complete rejection of the IM injected allogeneic BMSC transplant within 5 to 10 days. Immunosuppression with CsA could only marginally prolong graft survival. IM injected BMSC did not migrate to the site of the arterial ligation. CV injection of BMSC resulted in massive pulmonary infarction, leading to respiratory failure and death. Intrapulmonary cell trapping was evidenced by confocal endomicroscopy, BLI and fluorescence microscopy. IA injection of BMSC proved to be a feasible and safe strategy to bypass the lung circulation. During the follow-up period, neither BLI nor confocal endomicroscopy revealed any convincing ischemia-directed homing of BMSC.
BLI and confocal endomicroscopy are complementary imaging techniques for studying the in vivo biology of dual reporter gene-expressing BMSC. Allogeneic BMSC survival is limited in an immunocompetent host and cannot be preserved by CsA immunosuppression alone. We did not find substantial evidence for ischemia-directed BMSC homing and caution against CV injection of BMSC, which can lead to massive pulmonary infarction.
PMCID: PMC3534291  PMID: 23206380
Stem cell; BMSC; Homing; Bioluminescence; Confocal endomicroscopy
6.  Characterization of transferrin receptor-mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice (Oryza sativa L.) 
BMC Biotechnology  2012;12:92.
Transferrin (TF) plays a critical physiological role in cellular iron delivery via the transferrin receptor (TFR)-mediated endocytosis pathway in nearly all eukaryotic organisms. Human serum TF (hTF) is extensively used as an iron-delivery vehicle in various mammalian cell cultures for production of therapeutic proteins, and is also being explored for use as a drug carrier to treat a number of diseases by employing its unique TFR-mediated endocytosis pathway. With the increasing concerns over the risk of transmission of infectious pathogenic agents of human plasma-derived TF, recombinant hTF is preferred to use for these applications. Here, we carry out comparative studies of the TFR binding, TFR-mediated endocytosis and cellular iron delivery of recombinant hTF from rice (rhTF), and evaluate its suitability for biopharmaceutical applications.
Through a TFR competition binding affinity assay with HeLa human cervic carcinoma cells (CCL-2) and Caco-2 human colon carcinoma cells (HTB-37), we show that rhTF competes similarly as hTF to bind TFR, and both the TFR binding capacity and dissociation constant of rhTF are comparable to that of hTF. The endocytosis assay confirms that rhTF behaves similarly as hTF in the slow accumulation in enterocyte-like Caco-2 cells and the rapid recycling pathway in HeLa cells. The pulse-chase assay of rhTF in Caco-2 and HeLa cells further illustrates that rice-derived rhTF possesses the similar endocytosis and intracellular processing compared to hTF. The cell culture assays show that rhTF is functionally similar to hTF in the delivery of iron to two diverse mammalian cell lines, HL-60 human promyelocytic leukemia cells (CCL-240) and murine hybridoma cells derived from a Sp2/0-Ag14 myeloma fusion partner (HB-72), for supporting their proliferation, differentiation, and physiological function of antibody production.
The functional similarity between rice derived rhTF and native hTF in their cellular iron delivery, TFR binding, and TFR-mediated endocytosis and intracellular processing support that rice-derived rhTF can be used as a safe and animal-free alternative to serum hTF for bioprocessing and biopharmaceutical applications.
PMCID: PMC3521190  PMID: 23194296
Recombinant human serum transferrin; Transferrin receptor; Endocytosis; Cell growth and proliferation; Antibody production
7.  Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4) fusion protein 
BMC Biotechnology  2012;12:91.
The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR) is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv) which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S)4. In the present study, the anti-TfR single-chain antibody (TfRscFv) was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells.
Fusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM) analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA) assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4 complementary conjugation with the GAL4rec-GFP-pGes plasmid to verify the GLA4 activity and GAL4rec-recognized specificity functions. It also shows the complex, TfRscFv-GAL4-GAL4rec-GFP-pGes, could be taken into endochylema to express the green fluorescent protein (GFP) with 8 to 10-fold transfection efficiency.
Results of our study demonstrated that the biofunctianality of genetically engineered fusion protein, TfRscFv-GAL4, was retained, as the fusion protein could both carry the plasmid of GAL4rec-pGes and bind TfR on tumour cells. This product was able to transfect target cells effectively in an immuno-specific manner, resulting in transient gene expression. This protein that can be applied as an effective therapeutic and diagnostic delivery to the tumor using endogenous membrane transport system with potential widespread utility.
PMCID: PMC3560209  PMID: 23192001
8.  Chemo-enzymatic synthesis and in vitro cytokine profiling of tailor-made oligofructosides 
BMC Biotechnology  2012;12:90.
It is well known that carbohydrates play fundamental roles in cell signaling and infection processes as well as tumor formation and progression. However, the interaction pathways and cellular receptors targeted by carbohydrates and glycoconjugates remain poorly examined and understood. This lack of research stems, at least to a major part, from accessibility problems of large, branched oligosaccharides.
To test glycan - cell interactions in vitro, a variety of tailored oligosaccharides was synthesized chemo-enzymatically. Glycosyltransferases from the GRAS organisms Bacillus megaterium (SacB) and Aspergillus niger (Suc1) were used in this study. Substrate engineering of these glycosyltransferases generally acting on sucrose leads to the controlled formation of novel tailored di-, tri- and tetrasaccharides. Already industrially used as prebiotics in functional food, the immunogenic potential of novel oligosaccharides was characterized in this study. A differential secretion of CXCL8 and CCL2 was observed upon oligosaccharide co-cultivation with colorectal epithelial Caco-2 cells.
Pure carbohydrates are able to stimulate a cytokine response in human endothelial cells in vitro. The type and amount of cytokine secretion depends on the type of co-cultivated oligosaccharide.
PMCID: PMC3576278  PMID: 23181472
Oligofructoside; Glycosyltransferase; Suc1; Aspergillus niger; SacB; Bacillus megaterium; CXCL8 (IL-8); CCL2 (MCP-1); Caco-2
9.  Scalable production of biliverdin IXα by Escherichia coli 
BMC Biotechnology  2012;12:89.
Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. Biliverdin IXα, through interaction with biliverdin reductase, also initiates signaling pathways leading to anti-inflammatory responses and suppression of cellular pro-inflammatory events. The use of biliverdin IXα as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials. To address these limitations, methods to produce, recover and purify biliverdin IXα from bacterial cultures of Escherichia coli were investigated and developed.
Recombinant E. coli strains BL21(HO1) and BL21(mHO1) expressing cyanobacterial heme oxygenase gene ho1 and a sequence modified version (mho1) optimized for E. coli expression, respectively, were constructed and shown to produce biliverdin IXα in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXα than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXα production by strain BL21(mHO1) (up to an average of 23. 5mg L-1 culture) in fed-batch mode and production by strain BL21 (HO1) in batch-mode was scalable to 100L bioreactor culture volumes. Synthesis of the modified ho1 gene protein product was determined, and identity of the enzyme reaction product as biliverdin IXα was confirmed by spectroscopic and chromatographic analyses and its ability to serve as a substrate for human biliverdin reductase A.
Methods for the scalable production, recovery, and purification of biliverdin IXα by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXα and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.
PMCID: PMC3534565  PMID: 23176158
Biliverdin IXα; Heme oxygenase; Escherichia coli; HO1; Bilirubin; Anti-inflammatory; Biliverdin reductase; Bioreactor
10.  Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology 
BMC Biotechnology  2012;12:88.
An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein.
By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application.
In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
PMCID: PMC3526464  PMID: 23171216
11.  A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C 
BMC Biotechnology  2012;12:87.
Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.
An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.
This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.
PMCID: PMC3536577  PMID: 23153214
HIV-1; Clade C; V3; scFv
12.  Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease 
BMC Biotechnology  2012;12:86.
A systematic method for plant genome manipulation is a major aim of plant biotechnology. One approach to achieving this involves producing a double-strand DNA break at a genomic target site followed by the introduction or removal of DNA sequences by cellular DNA repair. Hence, a site-specific endonuclease capable of targeting double-strand breaks to unique locations in the plant genome is needed.
We engineered and tested a synthetic homing endonuclease, PB1, derived from the I-CreI endonuclease of Chlamydomonas reinhardtii, which was re-designed to recognize and cleave a newly specified DNA sequence. We demonstrate that an activity-optimized version of the PB1 endonuclease, under the control of a heat-inducible promoter, is capable of targeting DNA breaks to an introduced PB1 recognition site in the genome of Arabidopsis thaliana. We further demonstrate that this engineered endonuclease can very efficiently excise unwanted transgenic DNA, such as an herbicide resistance marker, from the genome when the marker gene is flanked by PB1 recognition sites. Interestingly, under certain conditions the repair of the DNA junctions resulted in a conservative pairing of recognition half sites to remove the intervening DNA and reconstitute a single functional recognition site.
These results establish parameters needed to use engineered homing endonucleases for the modification of endogenous loci in plant genomes.
PMCID: PMC3536558  PMID: 23148662
Homing endonuclease; I-CreI; Targeted marker excision
13.  A 28 nt long synthetic 5′UTR (synJ) as an enhancer of transgene expression in dicotyledonous plants 
BMC Biotechnology  2012;12:85.
A high level of transgene expression is required, in several applications of transgenic technology. While use of strong promoters has been the main focus in such instances, 5′UTRs have also been shown to enhance transgene expression. Here, we present a 28 nt long synthetic 5′UTR (synJ), which enhances gene expression in tobacco and cotton.
The influence of synJ on transgene expression was studied in callus cultures of cotton and different tissues of transgenic tobacco plants. The study was based on comparing the expression of reporter gene gus and gfp, with and without synJ as its 5′UTR. Mutations in synJ were also analyzed to identify the region important for enhancement. synJ, enhances gene expression by 10 to 50 fold in tobacco and cotton depending upon the tissue studied. This finding is based on the experiments comparing the expression of gus gene, encoding the synJ as 5′UTR under the control of 35S promoter with expression cassettes based on vectors like pBI121 or pRT100. Further, the enhancement was in most cases equivalent to that observed with the viral leader sequences known to enhance translation like Ω and AMV. In case of transformed cotton callus as well as in the roots of tobacco transgenic plants, the up-regulation mediated by synJ was much higher than that observed in the presence of both Ω as well as AMV. The enhancement mediated by synJ was found to be at the post-transcriptional level. The study also demonstrates the importance of a 5′UTR in realizing the full potential of the promoter strength. synJ has been utilized to design four cloning vectors: pGEN01, pBGEN02, pBGEN02-hpt and pBGEN02-ALSdm each of which can be used for cloning the desired transgene and achieving high level of expression in the resulting transgenic plants.
synJ, a synthetic 5′UTR, can enhance transgene expression under a strong promoter like 35S as well as under a weak promoter like nos in dicotyledonous plants. synJ can be incorporated as the 5′UTR of transgenes, especially in cases where high levels of expression is required. A set of vectors has also been designed to facilitate this process.
PMCID: PMC3536603  PMID: 23140609
Synthetic 5′UTR; Transgene expression; 35S promoter; Ω leader; AMV leader
14.  Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig 
BMC Biotechnology  2012;12:84.
Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for biomedical research. However, the efficiency of this approach is crucially dependent on the source of nuclear donor cells. In this study, we evaluate the potential of primary porcine kidney cells (PKCs) as cell source for SCNT, including their proliferation capacity, transfection efficiency, and capacity to support full term development of SCNT embryos after additive gene transfer or homologous recombination.
PKCs could be maintained in culture with stable karyotype for up to 71 passages, whereas porcine fetal fibroblasts (PFFs) and porcine ear fibroblasts (PEFs) could be hardly passaged more than 20 times. Compared with PFFs and PEFs, PKCs exhibited a higher proliferation rate and resulted in a 2-fold higher blastocyst rate after SCNT and in vitro cultivation. Among the four transfection methods tested with a GFP expression plasmid, best results were obtained with the NucleofectorTM technology, resulting in transfection efficiencies of 70% to 89% with high fluorescence intensity, low cytotoxicity, good cell proliferation, and almost no morphological signs of cell stress. Usage of genetically modified PKCs in SCNT resulted in approximately 150 piglets carrying at least one of 18 different transgenes. Several of those pigs originated from PKCs that underwent homologous recombination and antibiotic selection before SCNT.
The high proliferation capacity of PKCs facilitates the introduction of precise and complex genetic modifications in vitro. PKCs are thus a valuable cell source for the generation of porcine biomedical models by SCNT.
PMCID: PMC3537537  PMID: 23140586
Pig; Primary kidney cells; Fibroblasts; Nuclear transfer; Genetic engineering
15.  Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection 
BMC Biotechnology  2012;12:83.
There are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated.
In the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.
The data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.
PMCID: PMC3505465  PMID: 23134743
Apoptosis; Baculovirus; Chaperone; RNA interference; Sf-caspase-1
16.  Establishment of automated culture system for murine induced pluripotent stem cells 
BMC Biotechnology  2012;12:81.
Induced pluripotent stem (iPS) cells can differentiate into any cell type, which makes them an attractive resource in fields such as regenerative medicine, drug screening, or in vitro toxicology. The most important prerequisite for these industrial applications is stable supply and uniform quality of iPS cells. Variation in quality largely results from differences in handling skills between operators in laboratories. To minimize these differences, establishment of an automated iPS cell culture system is necessary.
We developed a standardized mouse iPS cell maintenance culture, using an automated cell culture system housed in a CO2 incubator commonly used in many laboratories. The iPS cells propagated in a chamber uniquely designed for automated culture and showed specific colony morphology, as for manual culture. A cell detachment device in the system passaged iPS cells automatically by dispersing colonies to single cells. In addition, iPS cells were passaged without any change in colony morphology or expression of undifferentiated stem cell markers during the 4 weeks of automated culture.
Our results show that use of this compact, automated cell culture system facilitates stable iPS cell culture without obvious effects on iPS cell pluripotency or colony-forming ability. The feasibility of iPS cell culture automation may greatly facilitate the use of this versatile cell source for a variety of biomedical applications.
PMCID: PMC3499150  PMID: 23127273
Induced pluripotent stem (iPS) cell; Automated cell culture system (ACCS); CO2 incubator-scale; Pluripotency
17.  NanoUPLC-MSE proteomic data assessment of soybean seeds using the Uniprot database 
BMC Biotechnology  2012;12:82.
Recombinant DNA technology has been extensively employed to generate a variety of products from genetically modified organisms (GMOs) over the last decade, and the development of technologies capable of analyzing these products is crucial to understanding gene expression patterns. Liquid chromatography coupled with mass spectrometry is a powerful tool for analyzing protein contents and possible expression modifications in GMOs. Specifically, the NanoUPLC-MSE technique provides rapid protein analyses of complex mixtures with supported steps for high sample throughput, identification and quantization using low sample quantities with outstanding repeatability. Here, we present an assessment of the peptide and protein identification and quantification of soybean seed EMBRAPA BR16 cultivar contents using NanoUPLC-MSE and provide a comparison to the theoretical tryptic digestion of soybean sequences from Uniprot database.
The NanoUPLC-MSE peptide analysis resulted in 3,400 identified peptides, 58% of which were identified to have no miscleavages. The experiment revealed that 13% of the peptides underwent in-source fragmentation, and 82% of the peptides were identified with a mass measurement accuracy of less than 5 ppm. More than 75% of the identified proteins have at least 10 matched peptides, 88% of the identified proteins have greater than 30% of coverage, and 87% of the identified proteins occur in all four replicates. 78% of the identified proteins correspond to all glycinin and beta-conglycinin chains.
The theoretical Uniprot peptide database has 723,749 entries, and 548,336 peptides have molecular weights of greater than 500 Da. Seed proteins represent 0.86% of the protein database entries. At the peptide level, trypsin-digested seed proteins represent only 0.3% of the theoretical Uniprot peptide database. A total of 22% of all database peptides have a pI value of less than 5, and 25% of them have a pI value between 5 and 8. Based on the detection range of typical NanoUPLC-MSE experiments, i.e., 500 to 5000 Da, 64 proteins will not be identified.
NanoUPLC-MSE experiments provide good protein coverage within a peptide error of 5 ppm and a wide MW detection range from 500 to 5000 Da. A second digestion enzyme should be used depending on the tissue or proteins to be analyzed. In the case of seed tissue, trypsin protein digestion results offer good databank coverage. The Uniprot database has many duplicate entries that may result in false protein homolog associations when using NanoUPLC-MSE analysis. The proteomic profile of the EMBRAPA BR-16 seed lacks certain described proteins relative to the profiles of transgenic soybeans reported in other works.
PMCID: PMC3532185  PMID: 23126227
Soybean; Seed proteomics; NanoUPLC-MSE; Uniprot database
18.  Antigen vehiculization particles based on the Z protein of Junin virus 
BMC Biotechnology  2012;12:80.
Arenavirus matrix protein Z plays an important role in virus budding and is able to generate enveloped virus-like-particles (VLPs) in absence of any other viral proteins. In these VLPs, Z protein is associated to the plasma membrane inner surface by its myristoyl residue. Budding induction and vesicle formation properties can be exploited to generate enveloped VLPs platform. These structures can be designed to carry specific antigen in the inner side or on the surface of VLPs.
Vaccines based on VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles in absence of viral nucleic acid, being noninfectious.
In this work we assayed the capacity of Junin Z protein to produce VLPs carrying the green fluorescent protein (eGFP), as a model antigen.
In this report the Junin Z protein ability to produce VLPs from 293T cells and its capacity to deliver a specific antigen (eGFP) fused to Z was evaluated. Confocal microscopy showed a particular membrane bending in cells expressing Z and a spot welded distribution in the cytoplasm. VLPs were detected by TEM (transmission electron microscopy) and were purified from cell supernatant. The proteinase protection assay demonstrated the VLPs integrity and the absence of degradation of the fused antigen, thus indicating its internal localization. Finally, immunization of mice with purified VLPs produced high titres of anti-eGFP antibodies compared to the controls.
It was proved that VLPs can be generated from cells transfected with a fusion Junin virus Z-eGFP protein in absence of any other viral protein, and the capacity of Z protein to support fusions at the C-terminal, without impairing its budding activity, allowing vehiculization of specific antigens into VLPs.
PMCID: PMC3534497  PMID: 23121996
Virus-like particles; Antigen delivery; Arenavirus; Z protein
19.  Live attenuated influenza viruses produced in a suspension process with avian AGE1.CR.pIX cells 
BMC Biotechnology  2012;12:79.
Current influenza vaccines are trivalent or quadrivalent inactivated split or subunit vaccines administered intramuscularly, or live attenuated influenza vaccines (LAIV) adapted to replicate at temperatures below body temperature and administered intranasally. Both vaccines are considered safe and efficient, but due to differences in specific properties may complement each other to ensure reliable vaccine coverage. By now, licensed LAIV are produced in embryonated chicken eggs. In the near future influenza vaccines for human use will also be available from adherent MDCK or Vero cell cultures, but a scalable suspension process may facilitate production and supply with vaccines.
We evaluated the production of cold-adapted human influenza virus strains in the duck suspension cell line AGE1.CR.pIX using a chemically-defined medium. One cold-adapted A (H1N1) and one cold-adapted B virus strain was tested, as well as the reference strain A/PR/8/34 (H1N1). It is shown that a medium exchange is not required for infection and that maximum virus titers are obtained for 1 × 10-6 trypsin units per cell. 1 L bioreactor cultivations showed that 4 × 106 cells/mL can be infected without a cell density effect achieving titers of 1 × 108 virions/mL after 24 h.
Overall, this study demonstrates that AGE1.CR.pIX cells support replication of LAIV strains in a chemically-defined medium using a simple process without medium exchanges. Moreover, the process is fast with peak titers obtained 24 h post infection and easily scalable to industrial volumes as neither microcarriers nor medium replacements are required.
PMCID: PMC3505166  PMID: 23110398
Live attenuated influenza virus; Influenza vaccine production; Suspension cell culture; Cell density effect; AGE1.CR.pIX
20.  Pandemic influenza vaccine: characterization of A/California/07/2009 (H1N1) recombinant hemagglutinin protein and insights into H1N1 antigen stability 
BMC Biotechnology  2012;12:77.
The recent H1N1 influenza pandemic illustrated the shortcomings of the vaccine manufacturing process. The A/California/07/2009 H1N1 pandemic influenza vaccine or A(H1N1)pdm09 was available late and in short supply as a result of delays in production caused by low yields and poor antigen stability. Recombinant technology offers the opportunity to shorten manufacturing time. A trivalent recombinant hemagglutinin (rHA) vaccine candidate for seasonal influenza produced using the baculovirus expression vector system (BEVS) was shown to be as effective and safe as egg-derived trivalent inactivated vaccine (TIV) in human clinical studies. In this study, we describe the characterization of the A/California/07/2009 rHA protein and compare the H1N1 pandemic rHA to other seasonal rHA proteins.
Our data show that, like other rHA proteins, purified A/California/07/2009 rHA forms multimeric rosette-like particles of 20–40 nm that are biologically active and immunogenic in mice as assayed by hemagglutination inhibition (HAI) antibody titers. However, proteolytic digest analysis revealed that A/California/07/2009 rHA is more susceptible to proteolytic degradation than rHA proteins derived from other seasonal influenza viruses. We identified a specific proteolytic site conserved across multiple hemagglutinin (HA) proteins that is likely more accessible in A/California/07/2009 HA, possibly as a result of differences in its protein structure, and may contribute to lower antigen stability.
We conclude that, similar to the recombinant seasonal influenza vaccine, recombinant A(H1N1)pdm09 vaccine is likely to perform comparably to licensed A(H1N1)pdm09 vaccines and could offer manufacturing advantages.
PMCID: PMC3538074  PMID: 23110350
Recombinant hemagglutinin; Influenza pandemic vaccine; H1N1; Baculovirus expression vector system (BEVS); Flublok; A(H1N1)pdm09
21.  Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver 
BMC Biotechnology  2012;12:78.
Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production.
To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629) encoding isopentenyl diphosphate isomerase (IPI) was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type) and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line) control.
Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our results demonstrated that regulation of IPI expression is a key target for efficient production of trans-polyisoprene in E. ulmoides.
PMCID: PMC3547716  PMID: 23110380
Isopentenyl diphosphate isomerase; Trans-polyisoprene; Natural rubber; Genetic transformation; Eucommia ulmoides
22.  Fluorescent bioassays for toxic metals in milk and yoghurt 
BMC Biotechnology  2012;12:76.
From a human health viewpoint, contaminated milk and its products could be a source of long-term exposure to toxic metals. Simple, inexpensive, and on-site assays would enable constant monitoring of their contents. Bioassays that can measure toxic metals in milk or yoghurt might reduce the risk. For this purpose, the green fluorescent protein (GFP)-tagged trans factors, ArsR-GFP and CadC-GFP, together with their cis elements were used to develop such bioassays.
ArsR-GFP or CadC-GFP, which binds either toxic metal or DNA fragment including cis element, was directly mixed with cow’s milk or yoghurt within a neutral pH range. The fluorescence of GFP, which is reflected by the association/dissociation ratio between cis element and trans factor, significantly changed with increasing externally added As (III) or Cd (II) whereas smaller responses to externally added Pb (II) and Zn (II) were found. Preparation and dilution of whey fraction at low pH were essential to intrinsic zinc quantification using CadC-GFP. Using the extraction procedure and bioassay, intrinsic Zn (II) concentrations ranging from 1.4 to 4.8 mg/l for milk brands and from 1.2 to 2.9 mg/kg for yoghurt brands were determined, which correlated to those determined using inductively coupled plasma atomic emission spectroscopy.
GFP-tagged bacterial trans factors and cis elements can work in the neutralized whole composition and diluted whey fraction of milk and yoghurt. The feature of regulatory elements is advantageous for establishment of simple and rapid assays of toxic metals in dairy products.
PMCID: PMC3505735  PMID: 23098077
23.  Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase 
BMC Biotechnology  2012;12:75.
Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.
In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.
Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.
PMCID: PMC3558336  PMID: 23092193
Cyathus bulleri; Heterologous laccase expression; Pichia pastoris; Recombinant laccase; Peptide mass fingerprinting
24.  Gene delivery to pancreatic exocrine cells in vivo and in vitro 
BMC Biotechnology  2012;12:74.
Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas.
For in vitro transfection/transduction of rat exocrine cells lipofection reagents, adenoviral vectors, and Mokola- and VSV-G pseudotyped lentiviral vectors were used. For in vivo transduction of mouse and rat pancreas adenoviral vectors and VSV-G lentiviral vectors were injected into the parenchymal tissue. Both lipofection of rat exocrine cell cultures and transduction with Mokola pseudotyped lentiviral vectors were inefficient and resulted in less than 4% EGFP expressing cells. Adenoviral transduction was highly efficient but its usefulness for gene delivery to rat exocrine cells in vitro was hampered by a drastic increase in cell death. In vitro transduction of rat exocrine cells was most optimal with VSV-G pseudotyped lentiviral vectors, with stable transgene expression, no significant effect on cell survival and about 40% transduced cells. In vivo, pancreatic cells could not be transduced by intra-parenchymal administration of lentiviral vectors in mouse and rat pancreas. However, a high efficiency could be obtained by adenoviral vectors, resulting in transient transduction of mainly exocrine acinar cells. Injection in immune-deficient animals diminished leukocyte infiltration and prolonged transgene expression.
In summary, our study remarkably demonstrates that transduction of pancreatic exocrine cells requires lentiviral vectors in vitro but adenoviral vectors in vivo.
PMCID: PMC3487942  PMID: 23088534
Lentiviral vector; Adenoviral vector; Lipofection; Gene transfer; Pancreas; Acinar cell
25.  Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells 
BMC Biotechnology  2012;12:72.
There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5′ flanking region of the mouse cirp gene.
By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5′ flanking region octanucleotide 5′-TCCCCGCC-3′ is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5′ flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5′ flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C.
Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.
PMCID: PMC3534229  PMID: 23046908
Cold shock protein; Stress response; Enhancer; Transcription factor; Recombinant protein

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