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PubMed1998

Effect of Insertion Site and Metabolic Load on the Environmental Fitness of a Genetically Modified Pseudomonas fluorescens Isolate.

An isolate of Pseudomonas fluorescens (SBW25) was modified with different marker genes (lacZY, aph-1, and xylE). These marker genes were inserted singly or in combination into two separate (1 Mbp apart) and presumably nonessential sites (-6- and Ee) on the chromosome of SBW25. This allowed the production of a range of genetically modified SBW25 variants that differed with respect to insertion site of the marker genes and metabolic burden. The environmental fitness of the different SBW25 variants was tested in soil, in the rhizosphere of wheat and pea, and on the phylloplane of wheat. Reduced environmental fitness of the different variants was mainly attributed to the extra metabolic burden of novel gene expression, whereas choice of insertion site was of little significance. Changes in environmental fitness were dependent on the environmental conditions; an environment, such as soil, with a low microbial carrying capacity had a negative effect on the environmental fitness of variants with a large metabolic load. In environments with a larger carrying capacity, such as the rhizosphere of pea, environmental fitness of variants with a large metabolic load was not significantly different from that of variants with a smaller metabolic burden.

Journal Article↗
PubMedDate not supplied

Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination.

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5+/-0.1 ng human NGF/ml/10(6) cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.

Animals↗
PubMed2003

Small-molecule-directed mpl signaling can complement growth factors to selectively expand genetically modified cord blood cells.

Efforts toward achieving gene therapy for blood disorders are plagued by low rates of gene transfer into hemopoietic stem cells. Recent studies suggest that this obstacle can be circumvented using selection. One way to achieve selection employs genes that encode receptor-bearing fusion proteins capable of inducing cell growth in response to drugs called chemical inducers of dimerization (CIDs). We have previously shown that genetically modified marrow cells from mice can proliferate for up to a year in culture in response to CID-initiated signals arising from the thrombopoietin receptor (mpl). The sustained growth observed in mouse hemopoietic cells results from an mpl-induced self-renewal of multipotential hemopoietic progenitor cells. In contrast, human hemopoietic cells proliferate only transiently in response to the mpl signal (from differentiation of transduced erythroid and megakaryocytic progenitors), while human myeloid progenitors fail to respond. Here, we show that myeloid progenitors from human cord blood can be induced to proliferate and/or differentiate in response to the mpl signal by providing additional signals via a combination of growth factors. These findings are relevant for the eventual clinical application of CID-regulated cell therapy.

Antigens, CD34↗
PubMed2001

Genetically modified Shiga toxin 2e (Stx2e) producing Escherichia coli is a vaccine candidate for porcine edema disease.

Porcine edema disease (ED) is an enterotoxaemia in pigs after weaning, caused by Shiga toxin 2e (Stx2e) producing Escherichia coli. Recently in Japan, outbreaks of ED are re-emerging in pig production. In this study we constructed a mutant that retained immunogenicity but lost Vero cell cytotoxicity, which produced genetically modified toxin (Stx2e*) by replacing glutamate with glutamine at position 167 and arginine with leucine at position 170 of the A subunit. The stx(2e)* gene was replaced with the stx(2e)gene of the wild type virulent strain by homologous recombination. As the parent wild strain was pathogenic to pigs but the mutant was not, the mutant named as YT106 was given to the pigs to examine its protective immunity against ED. All 20 pigs vaccinated with YT106 survived, but only eight of the 20 non-vaccinated pigs survived after the challenge with a wild strain. Also, the eight pigs that survived had decreased rates of gain relative to those of the controls. Blood IgG and intestinal IgA titres increased 3.3 and 1.6 times more than the control, respectively, showing that YT106 might be a good candidate of a live attenuated vaccine strain to protect against ED.

Animals↗
PubMed2000

Apoptosis induced by 5-flucytosine in human pancreatic cancer cells genetically modified to express cytosine deaminase.

AIM: To elucidate the pattern of 5-flucytosine (5-FC)-induced apoptosis and its role in gene therapy of human pancreatic cancer. METHODS: The human pancreatic cancer SW1990 cells (CEA-producing) were infected with recombinant adenoviruses (Adex1CEA-prCD or Adex1CEA-prZ). Expression of CD gene protein was examined by western blot. Apoptosis induced by 5-FC in human pancreatic cancer SW1990 cells genetically modified to express cytosine deaminase was observed by means of electron microscopy, DNA electrophoresis, and flow cytometry analysis techniques. RESULTS: The SW1990 cells infected with Adex1CEA-prCD were treated with 5-FC at 100 mumol.L-1 for 48 h, and cell apoptosis was observed. Typical apoptosis morphological feature appeared and DNA ladder could be demonstrated on DNA electrophoresis. Apoptosis peak was also showed by flow cytometry. Apoptotic cells accounted for 34.6% of the cell population. Cells in G1, S, and G2/M phase of cell cycle were 64%, 11%, and 7%, respectively. CONCLUSION: The apoptosis induced by 5-FC may be a primary mechanism in CD gene therapy of pancreatic cancer.

Adenoviruses, Human↗
PubMed2001

Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and growth regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an embryonic lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-1 constituents c-Jun and JunB, respectively, on human keratinocyte growth and differentiation. Furthermore, the hematopoietic growth factor granulocyte macrophage-colony stimulating factor has been identified as a novel regulator of keratinocyte growth and differentiation. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating factor and keratinocyte growth factor have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-1 by interleukin-1 released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and differentiation.

Adult↗
PubMed2005

Behavioral phenotypes of amyloid-based genetically modified mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative affliction of the elderly, presenting with progressive memory loss and dementia and terminating with death. There have been significant advances in understanding the biology and subsequent diagnosis of AD; however, the furious pace of research has not yet translated into a disease-modifying treatment. While scientific inquiry in AD is largely centered on identifying biological players and pathological mechanisms, the day-to-day realities of AD patients and their caregivers revolve around their steady and heartbreaking cognitive decline. In the past decade, AD research has been fundamentally transformed by the development of genetically modified animal models of amyloid-driven neurodegeneration. These important in vivo models not only replicate some of the hallmark pathology of the disease, such as plaque-like amyloid accumulations and astrocytic inflammation, but also some of the cognitive impairments relevant to AD. In this article, we will provide a detailed review of the behavioral and cognitive deficits present in several transgenic mouse models of AD and discuss their functional changes in response to experimental treatments.

Alzheimer Disease↗
PubMed2005

Delivery of therapeutic proteins to the mucosa using genetically modified microflora.

Drug delivery through mucosal surfaces offers a panorama of opportunities. The advantages are clear and include safety, ease of administration and higher social acceptance, although the major disadvantages are drug availability and appropriate drug targeting. Most mucosa are well equipped to manage the presence of bacteria and many are actually permanently colonised with a specific microflora. Such microbiota may become attractive tools for the delivery of a specific niche of protein therapeutics. These proteins can be produced from genetically modified microbes that are common to the mucosa, and their delivery to the host tissues has been demonstrated. This concept is being developed for the delivery of proteins to the intestine, but has also been applied in delivery to the vagina, nose and mouth.

Animals↗
PubMed2001

Pre-emptive targeting of the epitope spreading cascade with genetically modified regulatory T cells during autoimmune demyelinating disease.

Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th1-->Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.

Adoptive Transfer↗
PubMed2002

Insight into the physiological actions of thyroid hormone receptors from genetically modified mice.

Thyroid hormones exert a range of developmental and physiological actions in all vertebrates. Serum concentrations of L-thyroxine (T4) and 3,5,3 -L-triiodothyronine (T3) are maintained by a negative feedback loop involving T3-inhibition of hypothalamic thyrotrophin releasing hormone (TRH) and pituitary thyroid stimulating hormone (TSH) secretion, and by tissue specific and hormone-regulated expression of the three iodothyronine deiodinase enzymes that activate or metabolise thyroid hormones. T3 actions are mediated by two T3-receptors, TRalpha and TRbeta, which act as hormone-inducible transcription factors. The TRalpha (NR1A1) and TRbeta (NR1A2) genes encode mRNAs that are alternatively spliced to generate 9 mRNA isoforms (TRalpha1, alpha2, alpha3, Deltaalpha1, Deltaalpha2, beta1, beta2, beta3 and Deltabeta3), of which four (TRalpha1, alpha2, beta1 and beta2) are known to be expressed at the protein level in vivo. The numerous TR mRNAs are expressed widely in tissue- and developmental stage-specific patterns, although it is important to note that levels of mRNA expression may not correlate with receptor protein concentrations in individual tissues. The TRalpha2, alpha3, Deltaalpha1 and Deltaalpha2 transcripts encode proteins that fail to bind T3 in vitro. These non-binding isoforms, in addition to TRDeltabeta3 which does bind hormone, may act as dominant negative antagonists of the true T3-binding receptors in vitro, but their physiological functions and those of the TRbeta3 isoform have not been determined. In order to obtain a new understanding of the complexities of T3 action in vivo and the role of TRs during development, many mouse models of disrupted or augmented thyroid hormone signalling have been generated. The aim of this review is to provide a picture of the physiological actions of thyroid hormones by considering the phenotypes of these genetically modified mice.

Animals↗
PubMed2003

Delayed transplantation of fibroblasts genetically modified to secrete BDNF and NT-3 into a spinal cord injury site is associated with limited recovery of function.

Delivery of neurotrophic factors in acute models of spinal cord injury in adult rats can rescue axotomized neurons, promote axonal growth, and partially restore function. The extent to which repair and recovery of function can be achieved after chronic injury has received less attention. In the companion paper we show that transplanting fibroblasts genetically modified to produce neurotrophic factors into chronic (6-week) hemisection injuries results in sprouting, partial neuroprotection, but only limited regeneration. Here we describe functional consequences of this treatment using a series of behavioral tests. Adult rats received a complete unilateral C3/C4 hemisection and recovery from the injury was assessed over 5 weeks. At 6 weeks postoperative, the experimental group received grafts of a combination of fibroblasts modified to secrete BDNF or NT-3. The operated control groups received grafts of either gelfoam or gelfoam with fibroblasts expressing GFP into the lesion site. Behavioral recovery in the three groups was assessed over the next 10 weeks. Severe deficits with no recovery in any of the groups were observed in several tests (BBB, limb preference, narrow beam, horizontal rope test) that measure primarily motor function. Recovery was observed in the grid test, a measure of sensorimotor function, and the von Frey test, a measure of response to mechanical stimulation, but there were no differences between the operated control or experimental groups. Both groups also showed recovery from heat-induced hyperalgesia, with the experimental group exhibiting greater recovery than the operated control groups. In this test, delivery of neurotrophic factors from transplanted fibroblasts does not worsen responses to nociceptive stimuli and in fact appears to reduce hypersensitivity. Our data also demonstrate that additional damage to the spinal cord upon placement of a graft further compromises behavioral recovery for locomotor and postural function. Additional therapeutic interventions will be necessary to provide greater levels of recovery after chronic injuries.

Animals↗
PubMed2002

Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells.

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)

Animals↗
PubMed2004

Cellular immunotherapy for follicular lymphoma using genetically modified CD20-specific CD8+ cytotoxic T lymphocytes.

Humoral immunotherapy using the monoclonal anti-CD20 antibody rituximab induces remissions in approximately 60% of patients with relapsed follicular lymphoma; however, most patients eventually relapse despite continued expression of CD20 on lymphoma cells. We have hypothesized that cellular immunotherapy targeting CD20(+) cells might provide a more effective mechanism for eliminating lymphoma cells than anti-CD20 antibodies and are therefore investigating the utility of cytotoxic T lymphocytes (CTL) genetically modified to target the CD20 antigen. Peripheral blood mononuclear cells were activated with anti-CD3 antibody (OKT3) and recombinant human interleukin-2 and electroporated with a plasmid containing a CD20-specific scFvFc:zeta chimeric T cell receptor gene and a neomycin phosphotransferase gene (neo(R)). Transfected cells were selected using the antibiotic G418 and cloned by limiting dilution. Using this approach, we have generated CD8(+) CTL clones with CD20-specific cytotoxicity, which specifically lysed CD20(+) target cells, including actual tumor cells from patients with follicular lymphoma, small lymphocytic lymphoma, splenic marginal zone lymphoma, diffuse large B cell lymphoma, and chronic lymphocytic leukemia. The CTL clones have been expanded to numbers sufficient for therapy ( approximately 10(9) cells). Our data indicate the feasibility of generating and expanding CD20-specific CTL and, for the first time, demonstrate that such CTL exhibit specific cytotoxicity against actual tumor cells isolated from patients with a variety of B lymphoid malignancies. In view of these promising findings, a Phase I clinical trial for relapsed follicular lymphoma is being initiated.

Antigens, CD20↗
PubMed2001

Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo.

Human mesenchymal stem cells (MSCs) are capable of differentiating into multiple mesenchymal lineages including chondrocytes, osteocytes, adipocytes, and marrow stromal cells. Using a nonhuman primate model, we evaluated nonhuman primate MSCs as targets for gene therapy. Baboon MSCs (bMSCs) cultured from bone marrow aspirates appeared as a homogeneous population of spindle-shaped cells. bMSCs were capable of differentiating into adipocytes and osteocytes in vitro and chondrocytes in vivo. bMSCs were genetically modified with a bicistronic vector encoding the human erythropoietin (hEPO) gene and the green fluorescent protein (GFP) gene. Transduction efficiencies ranged from 72 to 99% after incubation of MSCs with retroviral supernatant. Transduced cells produced from 1.83 x 10(5) to 7.12 x 10(5) mIU of hEPO per 10(6) cells per 24 hr in vitro before implantation. To determine the capacity of bMSCs to express hEPO in vivo, transduced bMSCs were injected intramuscularly in NOD/SCID mice. In a separate experiment, transduced bMSCs were loaded into immunoisolatory devices (IIDs) and surgically implanted into either autologous or allogeneic baboon recipients. Human EPO was detected in the serum of NOD/SCID mice for up to 28 days and in the serum of five baboons for between 9 and 137 days. NOD/SCID mice experienced sharp rises in hematocrit after intramuscular injection of hEPO-transduced bMSCs. The baboon that expressed hEPO for 137 days experienced a statistically significant (p < 0.04) rise in its hematocrit. These data demonstrate that nonhuman primate MSCs can be engineered to deliver a secreted and biologically active gene product. Therefore, human MSCs may be an effective target for future human gene therapy trials.

Adult↗
PubMed2001

Effective treatment of established murine collagen-induced arthritis by systemic administration of dendritic cells genetically modified to express IL-4.

Dendritic cells (DC) are APCs that are able to stimulate or inhibit immune responses, depending on levels of expression of MHC class I and II costimulatory molecules and cytokines. Our previous studies have suggested that the observed contralateral effect, where injection of a vector carrying certain immunomodulatory genes into one joint resulted in inhibition of arthritis in untreated joints, is mediated by in vivo modification of DC. Therefore, we have examined the ability of genetically modified DC to suppress established murine collagen-induced arthritis (CIA) after i.v. delivery. IL-4 has been shown to partially reduce the severity of CIA after repeated injection of recombinant protein or by injection of an adenoviral vector expressing IL-4. Here we demonstrate that i.v. injection of immature DC, infected with an adenoviral vector expressing IL-4, into mice with established CIA resulted in almost complete suppression of disease, with no recurrence for up to 4 wk posttreatment. Injection i.v. of fluorescently labeled DC demonstrated that the cells rapidly migrated to the liver and spleen after 6 h and to the lymph nodes by 24 h. In culture, spleen cells from DC/IL-4-treated mice produced less IFN-gamma after stimulation by collagen than did control groups. In addition, DC/IL-4 administration decreased the level of specific Abs against type II collagen, in particular the IgG2 Th1 isotype 14 days posttreatment. These results demonstrate the ability to treat effectively established murine arthritis by systemic administration of DC expressing IL-4.

Adenoviridae↗
PubMed2006

Germline transmission of genetically modified primordial germ cells.

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.

Animals↗
PubMed2002

A preclinical model for the analysis of genetically modified human skin in vivo.

Although skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer, it is a challenging site when attempting gene delivery. In addition to the transience of gene expression, important obstacles to cutaneous gene therapy have included the inability to sustain gene expression in a large proportion of keratinocytes within a given skin compartment. In this study, we have developed a novel experimental strategy that allows long-term regeneration of entirely genetically engineered human skin on the backs of NOD/SCID mice. Primary human keratinocytes were infected with a retroviral vector encoding the enhanced green fluorescent protein (EGFP) produced by transient transfection of 293T cells. EGFP expression allowed cell-sorting selection of a polyclonal population of productively transduced keratinocytes that were assembled in a live fibroblast-containing fibrin dermal matrix and orthotopically grafted onto mice. Epifluorescent illumination of the transplanted zone allowed in vivo monitoring of the genetically modified graft. EGFP-positive human skin was present on mice for 22 weeks after grafting. In addition, frozen sections prepared from the grafts displayed consistently strong EGFP-based fluorescence in all epidermal strata at every time point examined. Persistence of transgene expression was further confirmed through EGFP protein immunodetection. Purified EGFP-positive keratinocytes grafted as part of the fibrin-based artificial skin were capable of generating multilayer human epidermis on mice, with well-developed granulosum and corneum strata, and clearly defined rete ridges. Finally, the large proportion of transduced keratinocytes in our grafts allowed us to study, for the first time, the long-term in vivo clonal reconstitution pattern of the regenerated skin. Analysis of the provirus insertion sites indicates that a discrete number of epidermal stem cell clones was responsible for the maintenance of human skin regenerated in NOD/SCID recipients.

Animals↗
PubMed2000

Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins.

Two enzymes, hydantoinase (HyuH) and L-N-carbamoylase (HyuC), are required for the biocatalytic production of natural and unnatural, optically pure L-amino acids starting from D,L-5-monosubstituted hydantoins using the so called 'hydantoinase-method'. For the preparation of immobilized enzymes, which omit several drawbacks of whole cell biocatalysts, purified or at least enriched HyuH and HyuC have to be provided. In order to simplify existing purification protocols several genetically modified derivatives of HyuH and HyuC from Arthrobacter aurescens DSM 3747 have been cloned and expressed in E. coli. A fusion protein consisting of maltose-binding protein (MalE) and HyuH resulted in an enhanced solubility of the hydantoinase, which easily forms inclusion bodies. On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose resin) and the complex purification protocol of the wild-type enzyme could therefore be simplified and shortened significantly. Interestingly, the specific activity of the MalE-HyuH fusion protein was as high as the wild-type enzyme despite that the molecular mass was doubled. A second modification of HyuH carrying a histidine-tag was efficiently bound to a metal affinity matrix but inactivated completely during elution from the column at either low pH or in the presence of imidazole. In the case of HyuC, an aspartate-tag has been added to the biocatalyst to allow an integrated purification-immobilization procedure since this enzyme is immobilized efficiently only via its carboxylic groups. The diminished isoelectric point of the Asp-tagged HyuC resulted in a simplified purification procedure. Compared to the wild-type enzyme expressed in E. coli HyuC-Asp6 was shifted off the elution range of the contaminating proteins and higher purification factors were obtained even in the capturing step. In contrast to HyuH, it was possible to purify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity using immobilized metal affinity chromatography. Therefore, the existing three step purification protocol was reduced to one chromatographic step and the yield of this relatively unstable protein enhanced remarkably.

Amidohydrolases↗