Staff publications

INTERVIEW

Angelo DePalma, Ph.D. (2013) Tying Up Loose Ends in Cell-Line Development. Genetic Engineering and Biotechnology News, Feb 1, 2013 (Vol. 33, No. 3), p 28-30. LINK

“Genome-editing technologies now permit precise positioning of deletions, modifications, and transgenes within living cells. These ideas have led Zsolt Keresztessy, Ph.D., senior research fellow at Proxencell, to a method employing sequence-specific meganucleases and TAL effector nucleases to generate stable monoclonal cell lines expressing membrane-bound antigens, FCGR receptors, and monoclonal antibodies. TAL effector nucleases are novel sequence-specific nucleases, formed by fusing a transcription activator-like (TAL) effector DNA binding domain to the catalytic head of an endonuclease…

…Dr. Keresztessy explains that specific genome editing technologies are still in the initial evolutionary phase—true especially for TAL effector nucleases. “That means, in addition to requiring substantial optimization work, investigators must also innovate in the adaptation of commercially available systems from, for example, Cellectis Bioresearch or Life Technologies.”Uncovering effective ways to transfer and express sequence-specific nucleases (e.g., plasmid DNA, mRNA, or proteins) into your target cells or cell lines, together with accessory sequences including like templates for homologous recombination or genome editing reporter constructs, is critical. “As a result,  we were forced to develop new technologies for assessing genome modifications at early stages of TAL transfections, strategies and tools for detecting and enriching knockout cells, and new approaches for mapping TAL specificity in vivo in automated and high-throughput assays.”…  >FULL ARTICLE

STAFF PUBLICATIONS

Keresztessy, Z. (2013) Genome Engineering Adventures in Cell Line Development: Reporters, effectors, producers. Informa Life Sciences: Cell Line Development and Engineering, 11 – 15 February 2013, NH Danube City, Vienna.

Recent specific genome editing technologies allow us to precisely position deletions, modifications, and transgenes in the genomes of living cells. Using various sequence-specific meganucleases and TAL effector nucleases engineered to recognise integrated target sequences or natural genomic loci of mammalian cells (Jurkat, HEK, YT, THP-1, CHO etc.), we have successfully generated a set of stable monoclonal cell lines expressing membrane-bound antigens, cell surface receptors, or monoclonal antibodies, to operate as target cells or effector cells in bioassays, or potential biopharmaceutical protein producers.

Keresztessy, Z., Attila Horváth, Ádám Pallér, László Steiner, József Horváth, Gábor Zahuczky, Endre Barta, Laszló Nagy, Bálint L. Bálint (2012) Developing immunoprecipitation/next generation sequencing-based technologies and tool for antibody characterisation: Specificity, batch-to-batch production QC, and full process controls for research, therapeutic, and diagnostic applications. PEGS Europe 2012, 6‐7 November 2012, Vienna. Austria.

Immunoprecipitation (IP) is a widespread method of purification of specific proteins (or co-purification of those with associated molecules  in Co-IP) from complex samples including cell lysates or whole tissue extracts. Chromatin immunoprecipitation (ChIP) is a method to study protein–DNA interactions, typically in-vivo interactions. The method combined with next generation sequencing (ChIP-NGS) is one of the most important functional genomic methods that had a significant impact on both gene regulation research and the field of epigenetics. One of the biggest limitations of the method is the lack of standards and controls providing clear results for the procedure. This generates significant variability and makes it difficult to introduce the method into clinical research. Here we aimed to develop novel approaches to standardise immunoprecipitation methods to monitor full processes up to the final analysis steps like NGS in ChIP-sec experiments. With the set of solutions in our hands, we not only offer (i) ways of robust standardisation and validation of clinical diagnostic protocols, but also (ii) highly sensitive analysis tools of antibody specificity down to molecular level, and (iii) quantitative approaches to antibody production QC, potentially with great impact on the development of  bispecific and next generation therapeutic antibodies.

Keresztessy, Z., Bálint, B.L., Zahuczky, G. and Nagy, L. (2011) Development of better antibody solutions for functional genomics and epigenetics. PEGS 2011 The Essential Protein Engineering Summit, Boston MA, USA. 9-13 May 2011.

In the field of functional genomics, there is a lack of reliable sources of antibodies and solutions for gene expression analyses based on chromatin immunoprecipitation techniques (ChIP, ChIP-Seq, and ChIP-on-Chip). The challanges antibody developers have to face include how to obtain antibodies capable of recognizing functional epitopes on proteins sequestered in large protein complexes regulating chromatin functions. Our major aim is to develop and optimize innovative antibody design algorythms, which are based on structural as well as functional information available on the target proteins, and involve integrated ensemble approaches to rational epitope prediction, to make possible the generation of highly target and application specific antibodies. Via the establishment of state of the art facilities in house, we produce polyclonal and monoclonal antibodies based on our rational epitopes, and the applicability and reliability of the resulting “better antibody solutions” are rigorously tested and characterised from a wide range of aspects including their effectivity in Western boltting, ELISA, immunoprecipitation, immunohystochemistry, mobility shift assays, ChIP, ChIP-Seq, ChIP-on-Chip applications, and quality controlled. Successful results demonstrated here are for cases of nuclear hormon receptors such as RXR and PPAR, and nuclear receptor co-activator/co-repressor complex members such as SMRT as our antibody targets, in comparison with commertially available counterparts.

 

Károly Jambrovics, Iván P. Uray, Zsolt Keressztesy, Jeffrey W. Keillor, László Fésüs, Zoltán Balajthy (2018) Transglutaminase 2 Programs Differentiating Acute Promyelocytic Leukemia Cells In All-Trans Retinoic Acid Treatment To Inflammatory Stage Through NF-KB Activation

Haematologica September 2018 : haematol.2018.192823; Doi:10.3324/haematol.2018.192823
http://www.haematologica.org/content/early/2018/09/28/haematol.2018.192823 (IF  9.096/2017)

Zoltan Simandi, Krisztian Pajer, Katalin Karolyi, Tatiana Sieler, Lu-Lin Jiang, Zsuzsanna Kolostyak, Zsanett Sari, Zoltan Fekecs, Attila Pap, Andreas Patsalos, Gerardo Alvarado Contreras, Balint Reho, Zoltan Papp, Xiufang Guo, Attila Horvath, Gréta Kiss, Zsolt Keresztessy, Gyorgy Vamosi, James Hickman, Huaxi Xu, Dorothee Dormann, Tibor Hortobagyi, Miklos Antal, Antal Nógrádi, and Laszlo Nagy. Arginine methyltransferase PRMT8 provides cellular stress tolerance in aging motoneurons. Jul 27, 2018  Journal of Neuroscience, Accepted Paper #JN-RM-3389-17R2 http://www.jneurosci.org/content/early/2018/07/27/JNEUROSCI.3389-17.2018 (IF  5.970.096/2017)

Jana Krenkova, Ákos Szekrényes, Zsolt Keresztessy, Frantisek Foret and  András Guttman (2013) Oriented Immobilization of Peptide-N-glycosidase F on a Monolithic Support for Glycosylation AnalysisJournal of Chromatography A , 1322, 54-61. (IF  4.6/2012) LINK

Király, R., Csősz, E., Kurtán, T., Antus, S., Szigeti, K., Vecsei, Z., Korponay-Szabó, I. R., Keresztessy, Z., and Fésüs, L. (2009) Functional significance of five non-canonical Ca2+-binding sites of transglutaminase 2 characterized by site directed mutagenesis. FEBS Journal, 276, 7083-7096. (IF 3.396 /2008) LINK

Keresztessy, Z., Bodnár, M., Ber, E., Hajdú, I., Zhang, M., Hartmann, J. F., Minko, T. and Borbély, J. (2009) Self-assembling chitosan/poly g-glutamic acid nanoparticles for targeted drug delivery. Colloid and Polymer Science 287, 759-765. (IF 1.736 / 2008) LINK

Keresztessy, Z., Csosz, E., Harsfalvi, J., Csomos, K., Gray, J., Lightowlers, R.N., Lakey, J.H., Balajthy, Z., and Fesus, L. (2006) Phage-Display Selection of Efficient Glutamine-Donor Substrate Sequences for Transglutaminase 2. Protein Science 15, 2466-2480. (IF 3.618 / 2005) LINK

Csosz, E., Keresztessy, Z., and Fesus, L. (2002) Transglutaminase substrates: from test tube experiments to living cells and tissues. Minerva Biotechnologica 14, 149-153. (IF 0.167 /2005)

Keresztessy, Z., Brown, K., Dunn, M.A., and Hughes, M.A. (2001) Identification of essential active-site residues in the cyanogenic b-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis. Biochemical Journal 353, 199-205. (IF 4.224 /2005) LINK

Ambrus, A., Bányai, I., Weiss, M.S., Hilgenfeld, R., Keresztessy, Z., Muszbek, L., and Fésüs, L. (2001) Calcium binding of transglutaminases: A 43Ca-NMR study combined with surface polarity analysis. J. Biomolecular Structure and Dynamics, 19, 59-74. (IF 1.430/2005) LINK

Nagy, Z., Keresztessy, Z., Szentirmai, A., Biró, S.(2001) Carbon source regulation of ß-galactosidase biosynthesis in Penicillium chrysogenum. Journal of Basic Microbiology 41, 351-362. (IF 1.000 /2005) LINK

Keresztessy, Z., and Hughes, M.A. (1998) Homology modelling of lipid-transfer proteins encoded by the barley low-temperature-inducible gene family blt4 and molecular dynamics aided analysis of fatty acid and lipid complexes. Plant Journal 14, 523-533. (IF 6.969 /2005) LINK

Hughes, J. Keresztessy, Z., Brown, K., Suhandono, S., and Hughes, M.A. (1998) Genomic organisation and srtucture of a-hydroxynitrile lyase in cassava (Manihot esculenta Crants). Archives of Biochemistry and Biophysics 356, 107-116. (IF 3.152/2005) LINK

Keresztessy, Z., Hughes, J., Kiss, L., and Hughes, M.A. (1996) Co-purification from E.coli of a plant b-glucosidase-GST fusion protein with the bacterial chaperonin GroEL. Biochemical Journal 314, 41-47. (IF 4.224 /2005) LINK

Keresztessy, Z., Kiss, L., and Hughes, M.A. (1994) Investigation of the active site of the cyanogenic b-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). II. Identification of Glu-198 as an active site carboxylate with acid catalytic function. Archives of Biochemistry and Biophysics 315, 323-330. (IF 3.152/2005) LINK

Hughes, M.A., and Hughes, J., Liddle, S., and Keresztessy, Z.: Biochemistry and molecular biology of cyanogenesis. Proc. Second Int. Meeting of the Cassava Biotechnology Network, August 20-27, 1994, Jakarta, Indonesia; CIAT Working Doc. No. 150, 385-395.

Keresztessy, Z., Kiss, L., and Hughes, M.A. (1994) Investigation of the active site of the cyanogenic b-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). I. Evidence for an essential carboxylate and a reactive histidine group in a single catalytic center. Archives of Biochemistry and Biophysics 314, 142-152. (IF 3.152/2005) LINK

Patents

Borbély, J., Bodnár, M., Hajdu, I., Hartmann, J.F., Keresztessy, Z., Nagy, L., Vamosi, G. Polymeric Nanoparticles by Ion-Ion Interactions. US Utility Patent Application No. US 60/833,672. PCT Patent Application No. WO/2009/035438. LINK