A. James Link
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 (609) 258-7191 |
 Engineering Quadrangle, A208, A403 |
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Lab (609) 258-4476 |
Research Focus
Nature has evolved and designed proteins to perform an exquisite array of tasks, but in the pursuit of biotechnological interests, these proteins must often be improved, altered, or even completely redesigned. In the post-genomic era, protein sequence information is abundant and readily available, and structural biology efforts are rapidly increasing the amount of protein structure information. However, the level of intricacy and complexity of most proteins is still such that rational design efforts are often unsuccessful in imparting an improved or new function to a protein. Fortunately, protein engineers can utilize an experimental algorithm that mimics Darwinian evolution to introduce new functions into proteins. In this algorithm, termed directed evolution, thousands or even millions of protein variants are generated by the introduction of mutations to the gene encoding the protein of interest. The library of protein variants is then screened to identify those members of the population with the highest levels of function or activity: a molecular survival of the fittest. One of the major focuses of the Link group is to apply directed evolution to medically relevant proteins.
Engineering high-affinity inhibitors of anti-apoptotic proteins
The molecular cause of several cancers is an imbalance between pro-apoptotic and anti-apoptotic proteins. It is postulated that the excess anti-apoptotic protein sequesters all of the pro-apoptotic protein thus preventing execution of apoptosis (programmed cell death). One potential treatment for cancers of this type is a high-affinity competitive inhibitor to the anti-apoptotic protein. We are pursuing several different natural proteins as scaffolds for such an inhibitor as well as completely de novo library designs.
Evolving higher efficacy antimicrobial peptides
Some classes of antimicrobial peptides are used by microbes as a defense mechanism against other species. These peptides represent an avenue of treatment for multidrug resistant (MDR) bacterial infections that has not been thoroughly explored yet. We will utilize naturally occurring antimicrobial peptides and apply directed evolution in order to engineer molecules with higher efficacies and broader spectra of activity. Synthetic biology principles are also being investigated in order to generate novel methods of delivering antimicrobial peptides to infection sites.
Biotechnological uses of E. coli: understanding the cellular response
Protein engineers often use host organisms such as E. coli simply as factories with little regard for the physiological state of the cell. We plan to use transcriptional reporters along with genomic and proteomic approaches (including BONCAT) to catalog the response of the cell to biotechnological uses of E. coli such as heterologous protein expression and unnatural amino acid incorporation. The knowledge and insights gained from these large-scale studies will subsequently be used to inform strain engineering experiments to develop host strains of E. coli that are more useful in biotech contexts.
Analyzing differential proteomes with BONCAT (Bio-Orthogonal Non-Canonical Amino Acid Tagging)
Selected Publications
Abdeljabbar DM, Song HJ, Link AJ: Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli. Biotechnology Letters 2011, in press.
Zhang SY, Link AJ: Bcl-2 Family Interactome Analysis Using Bacterial Surface Display. Integrative Biology 2011, 3: 823-831.
Abdeljabbar DM, Klein TJ, Link AJ: An Engineered Methionyl-tRNA Synthetase Enables Azidonorleucine Incorporation in Methionine Prototrophic Bacteria. ChemBioChem 2011, 12: 1699-1702.
Zhang SY, Prud'homme RK, Link AJ: Block Copolymer Nanoparticles as Nanobeads for the Polymerase Chain Reaction. Nano Letters 2011, 11: 1123-1126.
Pan SJ, Link AJ: Sequence Diversity in the Lasso Peptide Framework: Discovery of Functional Microcin J25 Variants with Multiple Amino Acid Substitutions. Journal of the American Chemical Society 2011, 133: 5016-5023.
Zhang SY, Adamson DH, Prud'homme RK, Link AJ: Photocrosslinking the Polystyrene Core of Block-Copolymer Nanoparticles. Polymer Chemistry 2011, 2: 665-671.
Pan SJ, Cheung WL, Fung HK, Floudas CA, Link AJ: Computational Design of Functional Variants of the Antimicrobial Peptide Microcin J25. Protein Engineering Design & Selection 2011, 24: 275-282.
Ferguson AL, Zhang SY, Dikiy I, Panagiotopoulos AZ, Debenedetti PG, Link AJ: An Experimental and Computational Investigation of Spontaneous Lasso Formation in Microcin J25. Biophysical Journal 2010, 99:3056-3065.
Mannoor MS, Zhang SY, Link AJ, McAlpine MC: Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides. Proceedings of the National Academy of Sciences, 107:19207-19212.
Cheung WL, Pan SJ, Link AJ: Much of the Microcin J25 Leader Peptide is Dispensable. Journal of the American Chemical Society 2010, 132:2514-2515.
Pan SJ, Cheung WL, Link AJ: Engineered Gene Clusters for the Production of the Antimicrobial Peptide Microcin J25. Protein Expression and Purification 2010, 71: 200-206.
Abdeljabbar DM, Klein TJ, Zhang SY, Link AJ: A Single Genomic Copy of an Engineered Methionyl-tRNA Synthetase Enables Robust Incorporation of Azidonorleucine into Recombinant Proteins in E. coli. Journal of the American Chemical Society 2009, 131:17078-17079.
Sun JJ, Abdeljabbar DM, Clarke N, Bellows ML, Floudas CA, Link AJ: Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface. Journal of Molecular Biology 2009, 394:297-305.
Tang Y, Wang P, Van Deventer JA, Link AJ, Tirrell DA: Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase. Chembiochem 2009, 10:2188-2190.
Xu LY, Link AJ: Stress responses to heterologous membrane protein expression in Escherichia coli. Biotechnology Letters 2009, 31:1775-1782.
Dieterich DC, Link AJ: Click Chemistry in Protein Engineering, Design, Detection and Profiling. in Click Chemistry in Biotechnology and Materials Science, Wiley, ed. Joerg Lahann.
Link AJ, Skretas G, Chari NS, Strauch EM, Georgiou, G: Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli Protein Science 2008, 17: 1857-1863.
Strable E, Prasuhn DE, Udit AK, Brown S, Link AJ, Ngo JT, Lander G, Quispe J, Potter CS, Carragher B, et al.: Unnatural amino acid incorporation into virus-like particles. Bioconjugate Chemistry 2008, 19:866-875.
Yoo TH, Link AJ, Tirrell DA: Evolution of a fluorinated green fluorescent protein. Proceedings of the National Academy of Sciences 2007, 104: 13887-13890.
Link AJ, Jeong KJ, Georgiou, G: Beyond toothpicks: new methods for isolating mutant bacteria. Nature Reviews Microbiology 2007, 5: 680-688.
Link AJ, Vink MKS, Tirrell DA: Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor. Nature Protocols 2007, 2: 1884-1887.
Link AJ, Vink MKS, Tirrell DA: Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer Nature Protocols 2007, 2: 1879-1883.
Link AJ, Georgiou G: Advances and Challenges in Membrane Protein Expression. AIChE Journal 2007, 53: 752-756.
Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM: Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nature Protocols 2007, 2: 532-540.
Link AJ, Vink MKS, Agard NJ, Prescher JA, Bertozzi CR, Tirrell DA: Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proceedings of the National Academy of Sciences 2006, 103: 10180-10185.
Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM: Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal non-canonical amino acid tagging (BONCAT). Proceedings of the National Academy of Sciences 2006, 103: 9482-9487.
Link AJ, Tirrell DA: Reassignment of Sense Codons In vivo. Methods 2005, 36: 291-298.
Link AJ, Vink MKS, Tirrell DA: Presentation and Detection of Azide Functionality in Bacterial Cell Surface Proteins. Journal of the American Chemical Society 2004, 126: 10598-10602.
Link AJ, Mock ML, Tirrell DA: Non-canonical Amino Acids in Protein Engineering. Current Opinion in Biotechnology 2003, 14: 603-609.
Link AJ, Tirrell DA: Cell Surface Labeling of Escherichia coli via Copper(I)- Catalyzed [3+2] Cycloaddition. Journal of the American Chemical Society 2003, 125:11164-11165.
Bradley AZ, Link AJ, Biswas K, Kahne D, Schwartz J, Jones M, Zhu ZD, Platz MS: Hydrogen Abstraction on Photolysis of a Naphthocarborane. Tetrahedron Letters 2000, 41: 8691-8694.
