American Society for Photobiology

ASP Conference 2016: 21-26 May 2016
Tampa Marriott Waterside Hotel & Marina


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6 - What is Photobiology? A Symposium for HS students

Florida 2   15:00 - 17:00

Chair(s): Frank Gasparro
 
6-1   What is Photobiology? An Introduction for High School Students. FP Gasparro*, Hamden Hall Country Day School

Abstract: This year a group of very talented Tampa area high school students will be attending our meeting for a day. They will be the Tampa Area Science Fair Finalists and they will be setting up their posters for our expert scientists to view. In addition, they have been invited to attend a special symposium in which several ASP members will be asked to present their work at a high school level. In this introductory talk, I will give an overview of the basics of photochemistry and photobiology so that the students will be familiar with some of the terminology and elementary principles. The primary purpose of this symposium is to introduce these talented students to the idea of conducting phootbiology research and thus to consider choosing photobiology as a field of study and career

6-2   Ultraviolet A - Induced Oxidation in Cornea : Characterization Of The Early Oxidation-Related Events C Zinflou*, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada ; PJ Rochette, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada

Abstract: Molecular and epidemiological data show that chronic exposure to sunlight ultraviolet (UV) rays, especially to UVA wavelengths (315 " 400 nm), is toxic for eyes and might be linked to many ocular diseases. UVA are the main component of solar UV reaching the eyes. Their absorption by cellular photosensitizers efficiently enhances oxygen reactive species production, leading to an oxidative stress induction. It has been described that UVA-induced oxidative stress plays a role in UVA toxicity. However, events triggered by oxidation and involved in the toxic effects of UVA radiations remain unknown. We have thus investigated the early cellular and molecular changes linked to oxidation in the cornea, the most UV-exposed ocular structure, following an ocular exposure to UVA rays. Three markers were used to assess changes throughout the three corneal layers (epithelium, stroma and endothelium) of UVA-irradiated (6000 kJ/m^2) rabbit eyes: (1) oxidized mitochondrial flavins/reduced NAD(P) (ox-Fvm/NAD(P)H) ratio as a sensor of mitochondrial activity and redox state; (2) 8-oxo-7,8-dihydroguanine (8-oxodG) formation in nuclei and mitochondria as a marker of photo-oxidative DNA damage and (3) levels of S-glutathionylated proteins (SG-Prot) as a marker of transiently impaired redox signaling. In response to UVA, we found significantly higher ox-Fvm/NAD(P)H ratio, reflective of a disrupted mitochondrial redox balance, in the most posterior parts of the cornea. Besides, UVA-induced 8-oxodG are concentrated in nuclear DNA of the epithelium, while they are found in both nuclear and mitochondrial DNA in stromal and endothelial layers. Finally, altered patterns of SG-Prot appear in the three layers immediately after UVA exposure and seem to correlate with UVA penetrance. Our data indicate that mitochondrial anomalies and prolonged disruption of proteins normal functions, under stress favored by a chronic UVA exposure, are events potentially critical for the development of UVA toxicity.

6-3   Rapid deamination of cyclobutane pyrmidine dimers at TCG sites in the FOS nucleosome in vivo VJ Cannistraro, Washington University ; S Pondugula, Washington University; Q Song, Washington University; JS Taylor*, Washington University

Abstract: C to T UV signature mutation hotspots in the p53 tumor suppressor gene of skin cancers occur primarily at methylated PyCpG sites that have been correlated with UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations can be explained by the rapid deamination of the C or 5-methyl-C in the CPD's to U or T followed by insertion of A by the DNA damage bypass polymerase η. As a consequence, the relative mutagenicity of C-containing CPDs is expected to depend on the relative deamination rates. In vivo, DNA is largely bound to nucleosomes which we have shown in vitro modulate the deamination rates of TmCG CPD's by 12-fold over a complete turn of the DNA helix. To determine the extent to which this occurs in vivo, we determined the deamination rates of CPD's at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells by LMPCR. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared to two other sites located at outside positions. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Most importantly, deamination of CPDs at TCG sites were faster than at all other C-containing dipyrimidine sites.

6-4   Hormonal regulation of the repair of UV photodamage in melanocytes by the MSH-MC1R signaling axis SG Jarrett, Univ. KY ; EM Wolf Horrell, Univ. KY; JA D'Orazio*, Univ. KY

Abstract: UV radiation represents a major causative risk factor for melanoma by promoting the formation of UV signature mutations. Nucleotide excision repair (NER), the genomic maintenance pathway responsible for clearing UV photodamage to prevent mutagenesis and malignant degeneration, is regulated hormonally by melanocyte stimulating hormone (MSH) and the melanocortin 1 receptor (MC1R). Loss-of-signaling MC1R polymorphisms that lead to defective cAMP second messenger generation are major inherited risk factors for melanoma development in part because of suboptimal NER and higher rates of UV mutagenesis. We have determined that the MC1R-cAMP signaling axis enhances NER through activation of cAMP-dependent protein kinase (PKA) and phosphorylation of ataxia telangiectasia and Rad3-related protein (ATR) on the S435 residue. This post-translational event recruits the key NER factor XPA to ATR and together, XPA and p-S435 ATR efficiently localize to sites of UV photodamage in chromatin to facilitate NER. The MC1R agonists melanocyte stimulating hormone (MSH) or adrenocorticotropic hormone (ACTH) efficiently promote generation of p-S435 ATR, accelerate repair of UV photoproducts and reduce UV mutagenesis. In contrast, MC1R antagonists agouti signaling protein (ASIP) or beta-defensin 3 (BD3) inhibit PKA-mediated ATR phosphorylation, impair NER and increase UV mutagenesis. Our data suggest that melanocytic NER is directly influenced by MC1R signaling ability znc and the presence of MC1R agonists and antagonists in the melanocytic milieu.

6-5   DNA repair genomics: Mapping DNA damage and DNA repair at single-nucleotide resolution across the human genome S Adar*, University of North Carolina ; J Hu, University of North Carolina; JD Lieb; A Sancar, University of North Carolina

Abstract: Ultraviolet radiation induces pyrimidine photodimers in DNA that present a barrier to transcription and replication, and compromise the ability of a cell to function. Nucleotide excision repair is the sole mechanism for removing these damages from the human genome. During human excision repair, dual incision of the damaged strand results in removal of a ~27 nucleotide-long single stranded oligomer. We have recently developed two genomic methods for mapping DNA damages and DNA repair at single nucleotide resolution across the human genome. Damages-seq relies on the replication-blocking properties of the damages to precisely map their location. In eXcision Repair-seq (XR-seq) we capture the excised oligonucleotide released during repair in vivo, and subject it to high-throughput sequencing. We used Damage-seq and XR-seq to produce genome-wide maps of two UV-induced DNA damages and follow the kinetics of their repair: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts ((6-4)PPs). Our results show preferential repair of actively transcribed and open chromatin regions. Conversely, repair at heterochromatic and repressed regions is relatively low and continues even two days following UV irradiation. Comparing repair kinetics with existing somatic mutation data from melanoma cancer cells shows late-repaired regions are associated with a higher level of cancer-linked somatic mutations. The new genomic assays we have developed will be a powerful tool in identifying key components of genome stability, and understanding the genetic and epigenetic changes resulting from genotoxic stress.

6-6   Tracking Photoinduced Charge Separation in DNA FD Lewis*, Northwestern University

Abstract: Recent studies of photoinduced charge separation in DNA have progressed rapidly as a consequence of collaborations with groups of laser spectroscopists and theoreticians. Recent studies of photoinduced charge separation in DNA have progressed rapidly as a consequence of collaborations with groups of laser spectroscopists and theoreticians. Old theories for the transport of positive charge (holes) and negative charge (electrons) founded on singlet step superexchange and multi-step incoherent hole hopping have been superseded by hole injection followed by formation of a conduction channel or trapped polaron. Direct evidence for formation of the trapped polaron in poly(adenine) sequences (A-tracks) of variable length has now been obtained by transient spectroscopy, as has the formation of the guanine hole trap. The efficiency of hole transport across an A-track is determined by the competition between charge recombination and charge transport in the polaron, both of which are subject to energy gap laws. Beyond five A-T base pairs the efficiency of charge separation becomes very small except when charge recombination is inhibited. Upon reaching a deep hole trap, the polaron is irreversibly trapped and decays slowly by charge recombination with the chromophore anion radical produced during the charge injection process. Electron transport is less efficient than is hole transport in DNA, because of faster charge recombination in the initially formed contact radical ion pair.

6-7   Molecular Mechanisms of Photosynthetic Antenna Regulation RE Blankenship*, Washington Univ.

Abstract: All photosynthetic organisms contain a light-harvesting antenna system. Photosynthetic antenna systems are extremely diverse in terms of structural organization and type of pigment utilized. In addition to the light absorption function of the antenna, it is essential for all photosynthetic organisms to have regulatory mechanisms that serve to protect them against excess light. Several of these regulatory mechanisms involve excited state quenching processes. These mechanisms are referred to as Non-Photochemical Quenching (NPQ) to distinguish them from the normal excited state quenching by photochemistry that leads to productive energy storage. Remarkably, there are several different types of NPQ that are distinct mechanistically and are almost certainly independent evolutionary inventions. This talk will center on two of the mechanisms of NPQ. These are the Orange Carotenoid Protein (OCP) that is found in photosynthetic cyanobacteria and the redox-induced quenching that has recently been discovered in the Fenna-Matthews-Olson (FMO) protein found in Green Sulfur Bacteria. Cyanobacterial OCP serves to regulate energy collection in the phycobilisome antenna complex, and contains a photoactivated 3'-hydroxyechinenone carotenoid molecule as the pigment, which is photoconverted from an orange form to a red form. While in the red form, the OCP binds to the phycobilisome and quenches excitations. The regulation in the FMO protein takes place via a pair of redox-active cysteine residues, which are near to two of the bacteriochlorophyll pigments. Under oxidizing conditions, these residues oxidize to form thiyl radicals, which directly quench the excited states of the nearby bacteriochlorophylls by electron transfer processes. These regulatory systems have been investigated using an interdisciplinary approach involving ultrafast spectroscopy, mass spectrometry, mutational analysis, molecular modeling, X-ray and neutron crystallography, EPR spectroscopy and electrochemistry.

6-8   Elucidation of the Photoprotective Mechanisms in Algal Light Harvesting G.S. Schlau-Cohen*, MIT

Abstract: In photosynthetic light harvesting, absorbed energy migrates through a protein network to reach a dedicated location for conversion to chemical energy. In green algae, this energy flow is efficient, directional, and regulated. The regulatory response involves complex and complicated multi-timescale processes that safely dissipate excess energy, thus protecting the system against deleterious photoproducts. We explore the mechanisms behind this photoprotective process in a light-harvesting complex implicated in dissipation, light-harvesting complex stress response (LHCSR). By characterizing the conformational states and dynamics of individual proteins, we identify the extent of energy dissipation in single LHCSR proteins and how the extent of dissipation changes in response to pH and carotenoid composition, two components known to play a role in photoprotection. From this information, we explore how individual complexes contribute to the balance between efficiency and adaptability in photosynthetic light harvesting.

6-9   Quantitative Imaging of Photosynthetic Pigments and Proteins in Live Cells JA Timlin*, Sandia National Laboratories

Abstract: In order to facilitate efficient energy harvesting and transfer, the endogenous pigments in photosynthetic organisms such as chlorophylls and carotenoids are collocated in pigment-protein complexes and have an inherently high degree of spectral overlap. Photosynthetic pigments and proteins vary within and between organisms and are dynamically regulated in response to changing environmental conditions. The identity, abundance, and localization of photosynthetic pigments are critical to understanding light harvesting and the reactions that produce energy within the cell. Spectral imaging methods coupled with multivariate analysis are uniquely suited to untangle the highly overlapped spectral signatures from photosynthetic pigments and reveal global pigment localization and dynamics in intact, living cells. In this talk, I will introduce the state-of-the-art in spectral imaging and multivariate analysis methods with an emphasis on preprocessing techniques that we have developed for robust analysis. Examples will highlight applications of hyperspectral confocal fluorescence microscopy and hyperspectral confocal Raman microscopy to identify and map multiple photosynthetic pigments in cyanobacteria, green algae, and land plants in response to changing conditions (environmental, genetic differences, etc.). These methods provide increased fundamental understanding of global pigment dynamics and function within and across photosynthetic organisms. The results have important implications for synthetic biology, genetic engineering, and development of biohybrid or bio-inspired devices.

6-10   Wavelengths from 385 " 405nm cause photodamage to skin cells KP Lawrence*, King's College London ; RPE Sarkany, King's College London; S Acker, BASF Grenzach GmbH; B Herzog, BASF Grenzach GmbH; AR Young, King's College London

Abstract: The adverse effects of solar UVR (~295 " 400nm) on the skin are well documented, especially in the UVB region (~295-315/320nm), and sunscreens have been shown to be beneficial in inhibiting a wide range of photodamage. The effects of long-wave UVA (>380nm) and visible radiation on the skin are much less well known, but increasingly studied. Most sunscreen formulations provide very little protection in the long wave UVA region (380-400nm) and almost none from shortwave visible wavelengths (400-420nm). We demonstrate photodamage in this region using high irradiance, narrowband LED arrays at 385nm and 405nm, using environmentally relevant doses. The endpoints include cell viability, DNA damage (cyclobutane pyrimidine dimers " CPD), differential gene expression and oxidative stress in vitro in HaCat keratinocytes, and pigmentation and erythema in vivo in human volunteers. For most endpoints we found a clear dose-response relationship for both sources. There was a highly significant reduction in cell viability (385nm p<0.0001; 405nm p<0.0001), increase in reactive oxygen species (385nm p<0.0001; 405nm p=0.0001), and several genes associated with adverse effects were significantly upregulated including HMOX-1, MMP-1, IL-1a, IL-6 and IL-8 (385nm p<0.02; 405nm p<0.003). PON-2 was upregulated by 385nm but not 405nm (385nm p=0.0002; 405nm p=0.1122). At high doses of 385nm radiation there was an increase in CPD production, but no effect at 405nm. In addition we demonstrate that these sources can induce skin-type dependent changes in erythema, IPD and delayed tanning in vivo. This work provides new insight into photodamage and may lead to new strategies to provide improved photoprotection in this poorly protected region.

6-11   Personalizing Photodynamic therapy based treatment strategies with Photoacoustic imaging S Mallidi*, Harvard Medical School ; M Ichikawa, Harvard Medical School; A Alkhateeb, Harvard Medical School; AP Khan, Harvard Medical School; Z Mai, Harvard Medical School; T Hasan, Harvard Medical School

Abstract: To achieve effective outcome in photodynamic therapy (PDT), it is paramount to understand the dynamic changes in the tumor microenvironment (oxygenation), photosensitizer consumption and adjust light dose accordingly. Towards this goal, I will present the utility of non-invasive 3D ultrasound guided photoacoustic imaging (PAI) to understand the heterogeneous changes in blood oxygen saturation during treatment and post treatment. Photoacoustic imaging, as the nomenclature suggests, involves acoustic signal generation by irradiating tissue with nanosecond laser pulses that satisfy the thermal stress confinement conditions. PAI provides tissue optical absorption information at deeper penetration depths with sensitivity similar to optical imaging and resolution on par with ultrasound imaging. Given that PAI and ultrasound imaging share the same receiver electronics, the images are inherently co-registered to provide both anatomical and tissue optical information. Leveraging the 3D imaging and real-time imaging capabilities of ultrasound guided PAI, we identify regions not responding to PDT and have the potential to recur using various subcutaneous and orthotopic mouse models. We further compare the predictive capability of photoacoustic imaging with the more predominantly used fluorescence imaging and immunohistochemistry techniques for both Benzoporphyrin derivative and aminolevulinic acid based PDT. Finally the strategies to push the envelop for ultrasound-guided PAI as an important aid in tumor diagnosis, customizing patient-specific treatment, and monitoring the therapeutic progression and outcome in vivo not only for PDT but other therapies will be discussed.

6-12   Informing and Implementing Combinational Approaches in Photodynamic Therapy TM Busch*, University of Pennsylvania

Abstract: Multimodality combinations of surgery, chemotherapy, molecular therapy, and radiotherapy play a major role in the treatment of cancer. Photodynamic therapy (PDT) can also uniquely contribute to cancer treatment in the combinational setting. The integration of PDT in a multimodality approach can be guided by pre-existing or therapy-altered characteristics of a tumor, such as its microenvironment or molecular signature. In this way, treatment can be rationally designed to synergize the anti-tumor effects of the separate modalities. However, in other circumstances, combined modality therapy is not designed. Rather, it is implicit to PDT delivery. For example, there is necessarily the combination of surgery with PDT when PDT is delivered intraoperatively to the site of tumor resection. Irrespective of whether combined therapy is designed or dictated, both the positive and negative interactions of the multiple therapies must be considered to guide clinical application. We discuss several combined modality approaches to PDT that are studied preclinically and clinically, considering the potential effects of each on the subsequent modality. In the case of intraoperative PDT, we've observed an effect of preceding surgery on the therapeutic potential of PDT that immediately follows. In combinations of PDT with molecular targeting drugs, we describe our recent data on the activation of epidermal growth factor receptor (EGFR) after high fluence rate PDT and consider fluence rate for its potential influence on the design of this multimodality approach. Ultimately, the elucidation and exploitation of interactions between PDT and other therapies will guide the design of new multi-modality treatments, as well as inform approaches to improve delivery of combinations that are already used clinically.

6-13   Light Effects on the Human Circadian Timing System JF Duffy*, Harvard Medical School and Brigham and Women's Hospital ; ED Chinoy, Harvard Medical School and Brigham and Women's Hospital; KM Zitting, Harvard Medical School and Brigham and Women's Hospital

Abstract: Nearly all organisms possess a biological timing system that produces rhythms in physiology and behavior with a ~24-hour cycle length. These near-24-h circadian rhythms are synchronized to the 24-h day by signals from the environment, and in humans this is primarily achieved by exposure to light and darkness. As a diurnal species, adult humans whose circadian system is synchronized with their environment will be alert during the day and able to sleep for an extended time during the night. The consequences of not being entrained are experienced by those such as shift workers who must remain awake at night to work and then attempt to sleep during the day, and by individuals who have recently traveled across multiple time zones. Light exposure has phase-dependent effects on circadian rhythms, with the magnitude and direction of alterations in rhythm timing dependent on the biological time at which the light exposure occurred. Other features including wavelength, illuminance, duration, and pattern of exposure also impact the circadian response to light. Thus, light exposure at some times of day will produce shifts in circadian rhythm timing to earlier hours, light at other times of day will produces shifts to later hours, and there are times at which the same duration, wavelength, and intensity of light will produce almost no change in circadian rhythm timing. In addition to the entraining and phase-shifting effects that light produces on human circadian rhythms, there are also direct effects, including suppression of the hormone melatonin and increases in alertness. With understanding of the direct and indirect light effects on the human circadian system, researchers have used that information to design therapies to improve on-shift alertness and off-shift sleep in night workers, to more quickly adjust the biological clocks of travelers, and to shift sleep timing in individuals with circadian rhythm sleep disorders.

6-14   Engineering Bioadhesive Biodegradable Nanoparticle Encapsulation of Organic Sunscreen Agents to Enhance Their Performance and Safety M Girardi*, Yale University ; Y Deng, Yale University; A Ediriwickrema, Yale University; J Lewis, Yale Unversity; HW Suh, Yale University; Linda Fong, Yale University; WM Saltzman, Yale University

Abstract: Skin cancer is the most common malignancy in the USA, and exposure to the sun's ultraviolet radiation (UVR) is the primary risk factor. Therefore, strategies designed to protect the skin from UVR exposure, including topically applied sunscreens, may markedly decreased the incidence of and costs associated with skin cancer. The ideal sunscreen formulation might be expected to provide all-day, broad UVA/UVB, waterproof/sweatproof protection from a single application, and as well appease any safety concerns by preventing penetration of the organic active ingredients into the skin cells and blood stream. Towards these goals, we have developed and are assessing the efficacy of encapsulation of organic sunscreen agents into biodegradable, bioadhesive nanoparticles (BNPs). BNPs were designed with a polylactic acid core linked to a hyperbranched polyglycerols corona that was terminated with aldehydes after exposure to sodium periodate. Upon application the skin, the BNPs form covalent bonding to the stratum corneum with uniform coverage. In pre-clinical testing, relative to commercially available sunscreen, BNP-encapsulated sunscreen showed superior substantivity, the capacity to prevent any detectable skin absorption of the active organic agent, and protection against UVR-induced cyclobutane pyrimidine dimer formation. Importantly, reactive oxygen species (ROS)-induced DNA damage following UVR exposure was clearly evident with commercial sunscreen, but owing to the prevention of penetration, absent with the BNP formulation. Thus, formulations utilizing BNP-encapsulation may enhance the performance and safety of the currently approved sunscreen agents.

6-15   Deleterious synergy between pollution and sunlight: pollutants from particulate matter aggravate oxidative impact of UVA1 in skin models. L MARROT*

Abstract: Atmospheric pollution is becoming a serious health concern in industrial countries and particulate matter (PM) from combustion is considered as particularly deleterious. In fact, ultrafine particles carry toxic compounds such as poly aromatic hydrocarbons (PAH). Moreover, they can translocate from lung capillaries to blood circulation and be distributed in the whole body. Up to now, no precise estimation of pollutants concentration in living skin is available. However, pollutants might reach dermis and epidermis either by penetration from skin surface or by systemic exposure. Some PAH are photo-reactive and phototoxic: sunlight and pollution might thus synergistically compromise skin health. After summing up current knowledge about dermatological damage induced by pollution, experimental data obtained in vitro using normal human keratinocytes or reconstructed epidermis will be presented. At very low concentrations (in the nanomolar range), some PAH such as benzopyrene or indenopyrene displayed a strong phototoxicity under UVA1 irradiation (340-400 nm). Even when cytotoxicity was low, PAH-induced photo-oxidative stress could impair mitochondrial function (membrane polarization and ATP production) and impact endogenous glutathione (GSH) homeostasis. Interestingly, among genes controlling GSH metabolism, SLC7A11 was particularly overexpressed (at gene and protein levels). This protein is an antiporter in charge of cystine supply. SLC7A11 upregulation suggests that regeneration of GSH might be of huge importance to ensure protection against "photo-pollution" stress. As proof, pretreatment of cells by buthionine sulfoximine BSO, an inhibitor of GSH biosynthesis, significantly increased PAH-induced phototoxicity. Our results highlight that pollutants could aggravate skin photodamage: specific photoprotection strategies for skin care in polluted area will be discussed.

6-16   Using Smartphone Hardware To Measure Ultraviolet Radiation. J Turner*, University of Southern Queensland ; AV Parisi, University of Southern Queensland; D Igoe, University of Southern Queensland; A Amar, University of Southern Queensland

Abstract: Smartphones are increasingly being used to provide people with the Ultraviolet Index (UVI) values and help them control their ultraviolet (UV) exposure. Various approaches have been applied to provide this service, some of which include external sensors and algorithm based prediction systems (Apps). Whilst these are excellent methods to improve education and understanding about UV exposure, one of the areas where the current products commercially available fail, (when no external sensor is available either due to availability or financially) is to account for areas where connectivity is intermittent or non-existent for an app that relies on network based information. In regional and remote areas of Australia, connectivity can still be an issue. In most UVI algorithm applications, the calculation of UVI is achieved from data collected in the nearest largest city that has the appropriate weather data collection systems. For some areas this could be an inappropriate estimation and provide incorrect UVI values. Additionally it fails to account for factors such as variation in cloud compared to location, and proximity of structures in the landscape. In order to determine UVI correctly, measurement of UVB radiation as well as UVA radiation is required. Previous work has shown it is possible to detect UVA radiation with existing hardware already contained within a smartphone (Igoe, Parisi & Carter, 2013). To measure UVI using existing smartphone hardware, UVB radiation must also be detectable within the same system. The feasibility of the use of smartphone hardware will be discussed and the smartphone's sensitivity to UVB radiation will be presented by considering data collected on the dark response, temperature response, irradiance response and spectral response for three different smartphone models. Discussion will also be included on the feasibility of the use of the smartphone as a UV measurement tool. Igoe, D., A. Parisi and B. Carter (2013)Photochem. Photobiol., vol. 89, pp. 215-218.

6-17   Purifying GFP By Modified Three-Phase Partitioning WW Ward*, Rutgers University ; C Turner, Rutgers University

Abstract: Three-phase partitioning (TPP) has been used by our group and others to purify green-fluorescent protein. TPP has reduced processing time from several months to one or two days. In purifying recombinant GFP from transformed E. coli cells, we have made three very useful modifications. We have switched from using t-butanol to the much less expensive isopropanol and we apply TPP directly to freshly harvested, un-lysed cells. Under the influence of 1.6 M ammonium sulfate, the alcohol behaves as a low polarity solvent, dissolving the cell membrane. The mixed solvent enters through the large cell wall pores and precipitates all DNA and most protein. The aggregated macromolecules become entombed within the cells while the more soluble GFP easily exits--usually 40% pure. We employ three precipitation stages rather than one or two. Then, one round of hydrophobic interaction chromatography increases purity to 90% or higher.



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