Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output

Loening, Andreas M.; Fenn, Timothy D.; Wu, Anna M.; Gambhir, Sanjiv S.

doi:10.1093/protein/gzl023

Cited by 386 publications

(364 citation statements)

References 35 publications

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“…Surface modification of the protein, Luc8, resulted in activity loss proportional to the degree of acryloylation; likely due to the fact that the lysine (which contain the free amines to be modified by) residues are important for Luc8 function [8]. On the other hand, if there is too little acryloylation, polymerization wouldn't happen and the stability improvement is minimal.…”

Section: Discussionmentioning

confidence: 99%

“…green fluorescent protein) in close proximity. In this study, QDs were covalently conjugated by EDC (N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride) coupling to the donor, Luc8 protein, an eight-mutation variant of the bioluminescent R. reniformis luciferase [8]. Upon the addition of substrate, coelenterazine, the protein emits blue light with a peak at 480 nm and this energy transfers to the QDs in a close proximity, causing QD fluorescence.…”

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confidence: 99%

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Improved QD-BRET conjugates for detection and imaging

Xing

Koh

et al. 2008

Biochemical and Biophysical Research Communications

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Self-illuminating quantum dots, also known as QD-BRET conjugates, are a new class of quantum dots bioconjugates which do not need external light for excitation. Instead, light emission relies on the bioluminescence resonance energy transfer from the attached Renilla luciferase enzyme, which emits light upon the oxidation of its substrate. QD-BRET combines the advantages of the QDs (such as superior brightness & photostability, tunable emission, multiplexing) as well as the high sensitivity of bioluminescence imaging, thus holds the promise for improved deep tissue in vivo imaging. Although studies have demonstrated the superior sensitivity and deep tissue imaging potential, the stability of the QD-BRET conjugates in biological environment needs to be improved for long-term imaging studies such as in vivo cell trafficking. In this study, we seek to improve the stability of QD-BRET probes through polymeric encapsulation with a polyacrylamide gel. Results show that encapsulation caused some activity loss, but significantly improved both the in vitro serum stability and in vivo stability when subcutaneously injected into the animal. Stable QD-BRET probes should further facilitate their applications for both in vitro testing as well as in vivo cell tracking studies. Keywordsself-illuminating quantum dots; bioluminescence resonance energy transfer (BRET); polymeric encapsulation; molecular imaging; nanotechnology Semiconductor quantum dots (QDs) have captivated scientists and engineers over the past two decades owing to their fascinating optical and electronic properties, which are not available from either single individual molecules or bulk solids [1]. Compared with organic dyes and fluorescent proteins, semiconductor QDs offer several unique advantages, such as size-and composition-tunable emission from visible to infrared wavelengths, large absorption coefficients across a wide spectral range, and very high levels of brightness and photostability [2]. These properties make them appealing fluorescent probes for bioimaging applications including in situ cell/tissue labeling, live cell imaging and in vivo imaging [3]. However, in vivo imaging using quantum dots still faces challenges such as interferences from tissue autofluorescence [4] and paucity of light available at non-superficial tissue sites [5]. One approach to solve these problems is to use high quality near infrared (NIR) QDs [6], which is an area still under active development. Alternatively, since both problems are related to the

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Improved QD-BRET conjugates for detection and imaging

Xing

Koh

et al. 2008

Biochemical and Biophysical Research Communications

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“…To address this 'self-illuminating' QDs have been developed which couple QDs with the Renilla luciferase. 89 By completely eliminating the need for excitation light, this very elegant approach could be refined by combining different luciferases with QDs of different wavelengths. 84 Overall, such strategies represent an exciting avenue for imaging bacteria within all tissue types which is ideally suited to cancer gene therapy-related applications.…”

Section: Instrumentation For Oimentioning

confidence: 99%

Bacterial vectors for imaging and cancer gene therapy: a review

et al. 2012

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The significant burden of resistance to conventional anticancer treatments in patients with advanced disease has prompted the need to explore alternative therapeutic strategies. The challenge for oncology researchers is to identify a therapy which is selective for tumors with limited toxicity to normal tissue. Engineered bacteria have the unique potential to overcome traditional therapies' limitations by specifically targeting tumors. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally, either external to (non-invasive species) or within tumor cells (pathogens). Pre-clinical and clinical investigations involving bacterial vectors require relevant means of monitoring vector trafficking and levels over time, and development of bacterial-specific real-time imaging modalities are key for successful development of clinical bacterial gene delivery. This review discusses the currently available imaging technologies and the progress to date exploiting these for monitoring of bacterial gene delivery in vivo.Cancer Gene Therapy ( INTRODUCTIONAlthough the potential anti-cancer effects of acquired bacterial infection have been evident for over 120 years and the presence of bacteria in excised tumors has been noted for over 60 years, 1 it is only in more recent times that the tumor-specific growth of bacteria has been considered for therapeutic purposes. While the precise mechanism(s) behind preferential bacterial tumor colonization remain poorly understood, this phenomenon must relate to unique tumor attributes that separate them from healthy tissues. Factors such as the irregular blood supply; local immune suppression; inflammation; and unique nutrient supply (e.g., purines) in tumors have been proposed to play a role. 2 Bacterial entry into the tumor environment is proposed to be facilitated by the leaky vasculature. Cancer cells are characterized by an aberrantly accelerated metabolism and proliferation leading to an imbalance of oxygen supply and consumption which is a major causative factor of tumor hypoxia. 3 The hypoxic nature of solid tumors enhances the growth of anaerobic and facultatively anaerobic bacteria. In addition, these areas with low oxygen and high interstitial pressure are considered to be an immunological sanctuary, where bacterial clearance mechanisms are greatly inhibited. 4 Necrotic regions are also rich in nutrients favoured by bacteria due to tumor cell turnover. However, tumor selective bacterial colonization now appears to be both bacterial species and tumor origin independent, since aerobic bacteria are also capable of colonising tumors, and even small tumors lacking an anaerobic center are colonised. See Figure 1 for proposed mechanisms.Invasive bacteria can internalize and replicate within tumor cells, while non-invasive bacteria grow externally to tumor cells, within the tumor micro-environment. 5 The tumor selective growth of bacteria has made them an attractive vehicle for the delivery of ...

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“…The SRL-PFAC system is advantageous because (a) the interaction of each Hsp90 isoform (a/h) with p23 can be individually monitored; (b) signal amplification is achieved through the enzymatic properties of complemented RL activity; (c) RL exhibits flash kinetics (25) and, thus, provides a real-time monitoring of Hsp90a/p23 and Hsp90h/p23 interactions; (d) SRL-PFAC has a high signal to background ratio and potentially less steric hindrance due to the small size of RL fragments (NRL, 23 kDa; CRL, 13 kDa); (e) RL does not require ATP for its activity, thus important for screening inhibitors that target the ATP-binding pocket of Hsp90; ( f ) the fusion constructs can be optimized and validated in cell culture before generation of stable cells; and (g ) repetitive imaging of the same mouse allows dynamic indirect monitoring of Hsp90a/p23 and Hsp90h/p23 interactions in response to treatment with Hsp90 inhibitors, wherein the pharmacokinetic properties of each Hsp90 inhibitor influence efficacy.…”

Section: Introductionmentioning

confidence: 99%

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

et al. 2008

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Heat shock protein 90A (Hsp90A)/p23 and Hsp90B/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90A/p23 and Hsp90B/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90A/p23 and Hsp90B/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90A/p23 and Hsp90B/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purinescaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90A/p23 and Hsp90B/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90A/p23 and Hsp90B/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoformselective Hsp90 inhibitors. [Cancer Res 2008;68(1):216-26]

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Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output

Cited by 386 publications

References 35 publications

Improved QD-BRET conjugates for detection and imaging

Improved QD-BRET conjugates for detection and imaging

Bacterial vectors for imaging and cancer gene therapy: a review

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

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