Engineering topics

Kiss, Csaba

Publications and source records attributed to Kiss, Csaba.

8 records

DOE OSTI2017

Method of generating ploynucleotides encoding enhanced folding variants

The invention provides directed evolution methods for improving the folding, solubility and stability (including thermostability) characteristics of polypeptides. In one aspect, the invention provides a method for generating folding and stability-enhanced variants of proteins, including but not limited to fluorescent proteins, chromophoric proteins and enzymes. In another aspect, the invention provides methods for generating thermostable variants of a target protein or polypeptide via an internal destabilization baiting strategy. Internally destabilization a protein of interest is achieved by inserting a heterologous, folding-destabilizing sequence (folding interference domain) within DNA encoding the protein of interest, evolving the protein sequences adjacent to the heterologous insertion to overcome the destabilization (using any number of mutagenesis methods), thereby creating a library of variants. The variants in the library are expressed, and those with enhanced folding characteristics selected.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Discovery of a Satellite of the Large Trans-Neptunian Object (225088) 2007 OR{sub 10}

2007 OR{sub 10} is currently the third largest known dwarf planet in the trans-Neptunian region, with an effective radiometric diameter of ∼1535 km. It has a slow rotation period of ∼45 hr that was suspected to be caused by tidal interactions with a satellite undetected at that time. Here, we report on the discovery of a likely moon of 2007 OR{sub 10}, identified on archival Hubble Space Telescope WFC3/UVIS system images. Although the satellite is detected at two epochs, this does not allow an unambiguous determination of the orbit and the orbital period. A feasible 1.5–5.8 · 10{sup 21} kg estimate for the system mass leads to a likely 35–100 day orbital period. The moon is about 4.ͫ2 fainter than 2007 OR{sub 10} in HST images that corresponds to a diameter of 237 km assuming equal albedos with the primary. Due to the relatively small size of the moon, the previous size and albedo estimates for the primary remains unchanged. With this discovery all trans-Neptunian objects larger than 1000 km are now known to harbor satellites, an important constraint for moon formation theories in the young solar system.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2016

LARGE SIZE AND SLOW ROTATION OF THE TRANS-NEPTUNIAN OBJECT (225088) 2007 OR{sub 10} DISCOVERED FROM HERSCHEL AND K2 OBSERVATIONS

We present the first comprehensive thermal and rotational analysis of the second most distant trans-Neptunian object (TNOs) (225088) 2007 OR{sub 10}. We combined optical light curves provided by the Kepler Space Telescope–K2 extended mission and thermal infrared data provided by the Herschel Space Observatory. We found that (225088) 2007 OR{sub 10} is likely to be larger and darker than derived by earlier studies: we obtained a diameter of d=1535{sub −225}{sup +75} km which places (225088) 2007 OR{sub 10} in the biggest top three TNOs. The corresponding visual geometric albedo is p{sub V}=0.089{sub −0.009}{sup +0.031}. The light-curve analysis revealed a slow rotation rate of P{sub rot} = 44.81 ± 0.37 hr, superseded by very few objects. The most likely light-curve solution is double-peaked with a slight asymmetry; however, we cannot safely rule out the possibility of having a rotation period of P{sub rot} = 22.40 ± 0.18 hr, which corresponds to a single-peaked solution. Due to the size and slow rotation, the shape of the object should be a MacLaurin ellipsoid, so the light variation should be caused by surface inhomogeneities. Its newly derived larger diameter also implies larger surface gravity and a more likely retention of volatiles—CH{sub 4}, CO, and N{sub 2}—on the surface.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2016

Evolution and characterization of a new reversibly photoswitching chromogenic protein, Dathail

In this paper, we report the engineering of a new reversibly switching chromogenic protein, Dathail. Dathail was evolved from the extremely thermostable fluorescent proteins thermal green protein (TGP) and eCGP123 using directed evolution and ratiometric sorting. Dathail has two spectrally distinct chromogenic states with low quantum yields, corresponding to absorbance in a ground state with a maximum at 389 nm, and a photo-induced metastable state with a maximum at 497 nm. In contrast to all previously described photoswitchable proteins, both spectral states of Dathail are non-fluorescent. The photo-induced chromogenic state of Dathail has a lifetime of ~ 50 min at 293 K and pH 7.5 as measured by UV–Vis spectrophotometry, returning to the ground state through thermal relaxation. X-ray crystallography provided structural insights supporting a change in conformation and coordination in the chromophore pocket as being responsible for Dathail's photoswitching. Neutron crystallography, carried out for the first time on a protein from the green fluorescent protein family, showed a distribution of hydrogen atoms revealing protonation of the chromophore 4-hydroxybenzyl group in the ground state. Additionally, the neutron structure also supports the hypothesis that the photo-induced proton transfer from the chromophore occurs through water-mediated proton relay into the bulk solvent. Beyond its spectroscopic curiosity, Dathail has several characteristics that are improvements for applications, including low background fluorescence, large spectral separation, rapid switching time, and the ability to switch many times. Therefore, Dathail is likely to be extremely useful in the quickly developing fields of imaging and biosensors, including photochromic Förster resonance energy transfer, high-resolution microscopy, and live tracking within the cell.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2014

THE ALBEDO-COLOR DIVERSITY OF TRANSNEPTUNIAN OBJECTS

We analyze albedo data obtained using the Herschel Space Observatory that reveal the existence of two distinct types of surface among midsized trans-Neptunian objects. A color-albedo diagram shows two large clusters of objects, one redder and higher albedo and another darker and more neutrally colored. Crucially, all objects in our sample located in dynamically stable orbits within the classical Kuiper Belt region and beyond are confined to the bright red group, implying a compositional link. Those objects are believed to have formed further from the Sun than the dark neutral bodies. This color-albedo separation is evidence for a compositional discontinuity in the young solar system.

79 ASTRONOMY AND ASTROPHYSICS↗

DOE OSTI2013

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes

Background: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. Methods: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. Results: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. Conclusions: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2013

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2013

Phanta: A Non-Fluorescent Photochromic Acceptor for pcFRET

We have developed an orange non-fluorescent photochromic protein (quantum yield, 0.003) we call Phanta that is useful as an acceptor in pcFRET applications. Phanta can be repeatedly inter-converted between the two absorbing states by alternate exposure to cyan and violet light. The absorption spectra of Phanta in one absorbing state shows excellent overlap with the emission spectra of a number of donor green fluorescent proteins including the commonly used EGFP. We show that the Phanta-EGFP FRET pair is suitable for monitoring the activation of caspase 3 in live cells using readily available instrumentation and a simple protocol that requires the acquisition of two donor emission images corresponding to Phanta in each of its photoswitched states. This the first report of a genetically encoded non-fluorescent acceptor for pcFRET.

59 BASIC BIOLOGICAL SCIENCES↗