Publications
118 results found
Goers L, Kylilis N, Tomazou M, et al., 2013, Engineering Microbial Biosensors, MICROBIAL SYNTHETIC BIOLOGY, Editors: Harwood, Wipat, Publisher: ELSEVIER ACADEMIC PRESS INC, Pages: 119-156, ISBN: 978-0-12-417029-2
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- Citations: 11
Polizzi KM, 2013, What Is Synthetic Biology?, SYNTHETI C BIOLOGY, Vol: 1073, Pages: 3-6, ISSN: 1064-3745
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- Citations: 6
Jimenez del Val I, Jedrzejewski PM, Exley K, et al., 2012, Application of Quality by Design paradigm to the manufacture of protein therapeutics, Glycosylation, Editors: Petrescu, ISBN: 980-953-307-130-0
Therapeutic glycoproteins represent one of the most important class products of the pharmaceutical industry, with 77 high-value drugs out of a total 642 approved by the European Medicines Agency. Their therapeutic efficacy, serum half-life and immunogenicity depends on the post-translational process of glycosylation, which is influenced by manufacturing process conditions. For this reason, protein glycosylation is a critical quality attribute for these drugs. Herein, we review the impact of glycosylation on product function, the role of manufacturing conditions in the resulting glycoform distribution and the current production systems employed industrially. We further present promising developments in terms of alternative, genetically engineered hosts, as well as advances in process operation that influence the glycan profile of the recombinant product. We finally present work on dynamic mathematical modeling for protein glycosylation that allows researchers to evaluate genetic engineering and process operation strategies in silico, with the aim of guiding experimentation. We demonstrate that such model-based approaches, when substantiated by experimental evidence, can support the Quality by Design initiative and expedite process development.
Polizzi KM, Isaacson RL, 2012, Protein construct optimization: data sharing strategy, PROTEIN & CELL, Vol: 3, Pages: 321-322, ISSN: 1674-800X
Sou SN, Ilieva KM, Polizzi KM, 2012, Binding of human BiP to the ER stress transducers IRE1 and PERK requires ATP, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol: 420, Pages: 473-478, ISSN: 0006-291X
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- Citations: 20
Behjousiar A, Kontoravdi C, Polizzi KM, 2012, In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture, PLOS One, Vol: 7
The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based) process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses.
Behjousiar A, Kontoravdi C, Polizzi KM, 2012, In situ monitoring of intracellular glucose and glutamine in CHO cell culture, PLoS One
Stefani IC, Wright D, Polizzi KM, et al., 2012, The role of ER stress-induced apoptosis in neurodegeneration, Current Alzheimer Research, Vol: 9, Pages: 373-387
Chen N, Koumpouras GC, Polizzi KM, et al., 2012, Genome-based kinetic modeling of cytosolic glucosemetabolism in industrially relevant cell lines -Saccharomyces cerevisiae and Chinese hamsterovary cells, Bioprocess and Biosystems Engineering, Vol: 35, Pages: 1023-1033
Song Y, Zhang D, Polizzi K, et al., 2012, Bacterial Whole Cell Bioreporters in Environmental Health, BIOSENSORS AND ENVIRONMENTAL HEALTH, Editors: Preedy, Patel, Publisher: CRC PRESS-TAYLOR & FRANCIS GROUP, Pages: 127-148, ISBN: 978-1-57808-735-8
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- Citations: 1
Chen N, Jimenez del Val I, Kyriakopoulos S, et al., 2011, Metabolic network reconstruction: advances in in silico interpretation of analytical information, Current Opinion in Biotechnology, Vol: 23, Pages: 1-6
Hallett J, Polizzi K, 2010, ORGANOSILICON-FUNCTIONAL PHASE TRANSFER CATALYSTS, US2010197892 (A1)
Organosilicon-functional phase transfer catalysts (PTCs) and methods for transferring immiscible molecules into a silicon-functional phase employing an organosilicon-functional PTC are provided.
Szita N, Polizzi K, Jaccard N, et al., 2010, Microfluidic approaches for systems and synthetic biology, CURRENT OPINION IN BIOTECHNOLOGY, Vol: 21, Pages: 517-523, ISSN: 0958-1669
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- Citations: 43
Polizzi K, Kontoravdi C, 2010, Vaccine production, Publisher: Taylor & Francis Group
Vaccines are biological preparations that improve an organism’s immunity to a particular disease. They can be prophylactic, such as influenza vaccines, or therapeutic, such as the vaccine Cervarix that is currently being administered to girls aged 12-13 in the U.K. to prevent cervical cancer due to infection with the human papilloma virus (HPV). Vaccines contain an agent that acts by stimulating the immune system to recognise this agent as a foreign entity. The immune system is then better prepared to recognise this agent and destroy it if the organism is infected with it in the future.
Chaparro-Riggers JF, Loo BLW, Polizzi KM, et al., 2007, Revealing biases inherent in recombination protocols, BMC Biotechnology, Vol: 7, Pages: 1-13, ISSN: 1472-6750
BackgroundThe recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.ResultsWe developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of β-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level).ConclusionBy comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an
Chaparro-Riggers JF, Rogers TA, Vazquez-Figueroa E, et al., 2007, Comparison of three enoate reductases and their potential use for biotransformations, ADVANCED SYNTHESIS & CATALYSIS, Vol: 349, Pages: 1521-1531, ISSN: 1615-4150
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- Citations: 99
Polizzi KM, Bommarius AS, Broering JM, et al., 2007, Stability of biocatalysts, CURRENT OPINION IN CHEMICAL BIOLOGY, Vol: 11, Pages: 220-225, ISSN: 1367-5931
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- Citations: 322
Chaparro-Riggers JF, Polizzi KM, Bommarius AS, 2007, Better library design: data-driven protein engineering., Biotechnol J, Vol: 2, Pages: 180-191
Data-driven protein engineering is increasingly used as an alternative to rational design and combinatorial engineering because it uses available knowledge to limit library size, while still allowing for the identification of unpredictable substitutions that lead to large effects. Recent advances in computational modeling and bioinformatics, as well as an increasing databank of experiments on functional variants, have led to new strategies to choose particular amino acid residues to vary in order to increase the chances of obtaining a variant protein with the desired property. Strategies for limiting diversity at each position, design of small sub-libraries, and the performance of scouting experiments, have also been developed or even automated, further reducing the library size.
Polizzi KM, Moore DA, Bommarius AS, 2007, A short-chain dehydrogenase/reductase from <i>Vibrio vulnificus</i> with both blue fluorescence and oxidoreductase activity, CHEMICAL COMMUNICATIONS, Pages: 1843-1845, ISSN: 1359-7345
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- Citations: 8
Bommarius AS, Broering JM, Chaparro-Riggers JF, et al., 2006, High-throughput screening for enhanced protein stability, CURRENT OPINION IN BIOTECHNOLOGY, Vol: 17, Pages: 606-610, ISSN: 0958-1669
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- Citations: 75
Polizzi KM, Parikh M, Spencer CU, et al., 2006, Pooling for improved screening of combinatorial libraries for directed evolution, BIOTECHNOLOGY PROGRESS, Vol: 22, Pages: 961-967, ISSN: 8756-7938
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- Citations: 23
Polizzi KM, Chaparro-Riggers JF, Vazquez-Figueroa E, et al., 2006, Structure-guided consensus approach to create a more thermostable penicillin G acylase., Biotechnol J, Vol: 1, Pages: 531-536
The thermostabilization of penicillin G acylase (PGA) is a difficult problem due to the large size of the protein and its complex maturation process. We developed a data-driven protein design method that requires fewer homologous sequences than the traditional consensus approach and utilizes structural information to limit the number of variants created. Approximately 50% of our 21 single-point mutants were found experimentally to be more thermostable than the wild-type PGA, two had almost threefold longer half-life at 50 degrees C, with very little effect on activity. An analysis of four programs that predict the thermostability conferred by point mutations shows little agreement between the programs and with the experimental data, emphasizing that the chosen stabilizing mutations are very difficult to predict, but that our data-driven design method should prove useful.
Bommarus AS, Polizzi KM, 2006, Novel biocatalysts: Recent developments, CHEMICAL ENGINEERING SCIENCE, Vol: 61, Pages: 1004-1016, ISSN: 0009-2509
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- Citations: 18
Polizzi KM, Spencer CU, Matsumura I, et al., 2005, Pooling of enzyme libraries for high throughput biocatalyst development
Biocatalysis is becoming increasingly important to the synthesis of pharmaceutical intermediates, aided by the ability to use directed evolution to tailor the specificity and stability of the biocatalyst for bioprocess optimization. Once a directed evolution library of enzymes is generated, a significant amount of effort is expended in high throughput screening to identify the improved mutants. Unfortunately, combinatorial bioengineering methods produce far more variants than most laboratories have the capacity to screen. Thus, the discovery of the best mutants becomes driven by the probability of sampling these from the screening pool. Pooling multiple mutants into each assay well increases the accessible sequence space, but must be balanced by the detection limit of the assay. We have mapped out the conditions for effective pooling, particularly with regard to assay accuracy, the fraction of good mutants created, and their activity level improvement. First, we developed Monte-Carlo simulation models of pooling and experimentally validated them using progressively complex scenarios, ranging from mixing just a rarely occurring "supermutant" in low frequency with an ancestral enzyme to the screening of an actual directed evolution library. In each case, the number of "supermutants" detected by the simulation model and in the experiment was nearly identical. Pooling increases the chances of detecting the "supermutant" in all cases, regardless of assay accuracy. Subsequently, using pooling to evolve β-galactosidase into a β-fucosidase, we verified the effectiveness of pooling over a conventional evolution approach.
Polizzi KM, Chaparro-Riggers J, Loo B, et al., 2005, Increasing the synthetic utility of penicillin G acylase by rational and directed evolution
Penicillin G acylase (PGA) is an important enzyme in the semi-synthetic production of β-lactam antibiotics. Despite many years of research, the enzyme is still lacking in stability and substrate specificity. Improvements to these properties would increase the utility of the enzyme and decrease the cost of antibiotic production. We set out both to improve the thermostability and to improve the kinetics of PGA. The PGA enzyme is a large, hetereodimeric enzyme which is difficult to evolve using truly random methods. Therefore, we began our efforts with an extensive bioinformatic analysis in an effort to limit the search space for protein engineering. For improvement of thermostability, we targeted specific residues using a consensus-based sequence comparison approach. Additional work focuses on using directed evolution to improve the synthetic utility of the enzyme by mutagenesis targeted to the segments of the α- and β- chains that are in contact with the substrate binding pocket. To facilitate directed evolution, we have obtained and characterized the kinetics of the penicillin G acylase genes from 4 different organisms, each with different stabilities and catalytic characteristics. In the presentation, we will cover both the approach as well as the results to date.
Polizzi KM, Spencer CU, Dubey A, et al., 2005, Simulation modeling of pooling for combinatorial protein engineering., J Biomol Screen, Vol: 10, Pages: 856-864, ISSN: 1087-0571
Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, betagalactosidase (supermutant, or greatly improved enzyme) and beta-glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Additional simulations using more complex activity distributions show the versatility of the model. Pooling is most suited to cases such as the directed evolution of new function in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling is most successful when a sensitive assay is employed. Using the model will increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.
Polizzi KM, Spencer CU, Dubey A, et al., 2005, Simulation modeling of pooling for combinatorial protein engineering, JOURNAL OF BIOMOLECULAR SCREENING, Vol: 10, Pages: 856-864, ISSN: 1087-0571
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- Citations: 8
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