We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. Several approaches are being developed to image reporter gene expression in living animals. These include methods that rely on charge-coupled device camera imaging and bioluminescent reporter genes (1), single-photon emission computed tomography using the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene (2), approaches that use magnetic resonance imaging (reviewed in ref.3), methods based on the HSV1-tk reporter gene and positron emission tomography (PET) (4, 5), and the use of the dopamine type 2 receptor (D2R) as a reporter gene for PET (6). The use of reporter genes that can be imaged in vivo will permit many different applications, including monitoring of both somatic gene transfer and transgenic͞knock-in reporter gene expression (7).PET provides repeated, noninvasive imaging of biological processes in living subjects (8, 9). PET utilizes molecular probes labeled with positron-emitting radioisotopes (e.g., fluorine-18, with a half-life of 110 min). PET probes typically are either positron-labeled ligands for receptors or positron-labeled substrates for intracellular enzymes. Tracer quantities of PET probes yield a tomographic image after their retention, as a consequence of either binding of positron-labeled ligand to a receptor or conversion of positron-labeled substrate to ''trapped'' metabolic product(s). PET is particularly well suited for application to human studies. PET reporter gene imaging in humans will allow monitoring of the location(s), magnitude, and duration of therapeutic͞suicide gene expression, by using vectors for DNA delivery in which the reporter gene and therapeutic gene are expressed from a common transcript (10,11 HSV1-tk refers to the gene, HSV1-TK refers to the enzyme.) HSV1-TK phosphorylates a range of substrates, including acycloguanosines (e.g., acyclovir, ganciclovir, penciclovir) and uracil derivatives [e.g., 2Ј-f luoro-2Ј-deoxy-1--arabinofuranosyl-5-iodouracil (FIAU)]. In contrast, mammalian thymidine kinases phosphorylate acycloguanosines only minimally, making these substrates advantageous as reporter gene imaging probes (11). Acycloguanosine derivatives are currently used extensively both as cytotoxic pharmaceuticals to treat herpes infections and for HSV1-tk suicide gene therapy (13).Improvements in sensitivity of the HSV1-tk reporter gene imaging assay can be achieved either (i) by identifying substrates that exhibit higher V max ͞K m for HSV1-TK or (ii) by engineering TK enzyme(s) with improved V max ͞K m for a particular reporter substrate. Decreased V max ͞K m of HSV1-TK for thymidine (an endogenous competitor) also should improve HSV1-tk reporter gene assay sensiti...
The thermostabilization of an enzyme while maintaining its activity for industrial or biomedical applications using traditional selection methods can be difficult. We demonstrate a rapid computational approach that identified three mutations within a model enzyme that produce a 10°C increase in apparent T m and a 30-fold increase in half-life at 50°C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induces an increased, temperaturedependent bacterial growth rate under conditions that require its activity, thereby coupling molecular and metabolic engineering.Enzymes are the most efficient catalysts of chemical reactions known, enhancing reaction rates by as much as twenty-three orders of magnitude (1, 2). However, there has been little evolutionary pressure for them to become more thermostable than is required by their native environment. Many studies indicate that enzymes (like most proteins) exhibit closely balanced free energy profiles for folding and unfolding, thereby allowing functionally important dynamic motions and appropriate degradation in vivo (3). However, in a laboratory or industrial setting this lack of thermostability can lead to undesirable loss of activity (4).The physical principles of protein folding that result in a balance of stability and flexibility, while maintaining function, are not perfectly understood and have been difficult to exploit for the development of thermostabilized enzymes (4). For hyperthermophiles, selective pressures have generated proteins with denaturation temperatures upwards of 110° C (5). Their proteins exhibit topologies and stabilizing interactions similar to those from mesophilic and thermophilic organisms (6, 7) leading to diverse hypotheses regarding their relative behaviors (8). However, a key mechanism for thermostabilization appears to be optimization of interactions between amino acids within their core (5), complementing computational design methods which optimize a sequence for a given fold (9-13).The thermostabilization of an enzyme presents additional challenges for computational protein design methods because the active site substrate geometry and the molecular dynamic behavior during an enzymatic reaction often appear fine-tuned for maximum catalytic efficiency (2, 3). Therefore the design method must be capable of predicting thermostabilizing mutations within a given fold while minimizing any shift in the backbone that might structurally disrupt the active site structure or quench its flexibility. In the past several years, methods for computational protein structure prediction and design have improved significantly (10,11,14). Recently, computational design has been used successfully in thermostabilizing non-catalytic proteins (15-18), redesigning binding pockets (19)(20)(21)(22)(23), creating a novel protein fold (24) and designing catalytic activity into a bacterial receptor (25). We use the program RosettaDe...
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