A. James Link
Engineering Quadrangle, A208, A403
Lab (609) 258-4476
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)
Maksimov MO, Koos JD, Zong C, Lisko B, Link AJ. (2015) Elucidating the specificity determinants of the AtxE2 Lasso Peptide Isopeptidase. J Biol Chem. [Epub ahead of print]
Zong C, Maksimov MO, Link AJ. (2015)Construction of Lasso Peptide Fusion Proteins. ACS Chem Biol. [Epub ahead of print]
Link AJ. (2015) Biosynthesis: Leading the way to RiPPs. Nat Chem Biol. 11(8): 551-2. Pubmed
Futran AS, Kyin S, Shvartsman SY, Link AJ. (2015) Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking. Proc Natl Acad Sci U S A. [Epub ahead of print]
Piscotta FJ, Tharp JM, Liu WR, Link AJ. (2015) Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids. Chem Commun (Camb). 51: 409-12. Pubmed
Futran AS, Link AJ, Seger R, Shvartsman SY. (2013) ERK as a model for systems biology of enzyme kinetics in cells. Curr Biol. 23: R972-79. Pubmed
Maksimov MO, Link AJ. (2013) Prospecting genomes for lasso peptides. J Ind Microbiol Biotechnol. 41: 333-44. Pubmed
Maksimov MO, Link AJ. (2013) Discovery and characterization of an isopeptidase that linearizes lasso peptides. J Am Chem Soc. 135: 12038-47. Pubmed
Maksimov MO, Pelczer I, Link AJ. (2012) Precursor-centric genome-mining approach for lasso peptide discovery. Proc Natl Acad Sci. 109: 15223-28. Pubmed
Zhang S, Long A, Link AJ. (2012) A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins. ACS Synth Biol. 1: 89-98. Pubmed
Abdeljabbar DM, Song HJ, Link AJ. (2012) Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli. Biotechnol Lett. 34: 91-96. Pubmed
Zhang S, Link AJ. (2011) Bcl-2 family interactome analysis using bacterial surface display. Integr Biol (Camb). 3: 823-31. Pubmed
Abdeljabbar DM, Klein TJ, Link AJ. (2011) An engineered methionyl-tRNA synthetase enables azidonorleucine incorporation in methionine prototrophic bacteria. Chembiochem. 12: 1699-702. Pubmed
Zhang S, Prud'homme RK, Link AJ. (2011) Block copolymer nanoparticles as nanobeads for the polymerase chain reaction. Nano Lett. 11: 1723-26. Pubmed
Pan SJ, Link AJ. (2011) Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions. J Am Chem Soc. 133: 5016-23. Pubmed
Zhang SY, Adamson DH, Prud'homme RK, Link AJ. (2011) Photocrosslinking the polystyrene core of block-copolymer nanoparticles. Polymer Chem. 2: 665-71.
Pan SJ, Cheung WL, Fung HK, Floudas CA, Link AJ. (2011) Computational design of the lasso peptide antibiotic microcin J25. Protein Eng Des Sel. 24: 275-82. Pubmed
Ferguson AL, Zhang S, Dikiy I, Panagiotopoulos AZ, Debenedetti PG, Link AJ. (2010) An experimental and computational investigation of spontaneous lasso formation in microcin J25. Biophys J. 99: 3056-65. Pubmed
Mannoor MS, Zhang S, Link AJ, McAlpine MC. (2010) Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides. Proc Natl Acad Sci. 107: 19207-12. PubMed
Cheung WL, Pan SJ, Link AJ. (2010) Much of the microcin J25 leader peptide is dispensable. J Am Chem Soc. 132: 2514-15. Pubmed
Pan SJ, Cheung WL, Link AJ. (2010) Engineered gene clusters for the production of the antimicrobial peptide microcin J25. Protein Expr Purif. 71: 200-06. Pubmed
Abdeljabbar DM, Klein TJ, Zhang S, Link AJ. (2009) A single genomic copy of an engineered methionyl-tRNA synthetase enables robust incorporation of azidonorleucine into recombinant proteins in E. coli. J Am Chem Soc. 131: 17078-79. Pubmed
Sun J, Abdeljabbar DM, Clarke N, Bellows ML, Floudas CA, Link AJ. (2009) Reconstitution and engineering of apoptotic protein interactions on the bacterial cell surface. J Mol Biol. 394: 297-305. Pubmed
Tang Y, Wang P, Van Deventer JA, Link AJ, Tirrell DA. (2009) Introduction of an aliphatic ketone into recombinant proteins in a bacterial strain that overexpresses an editing-impaired leucyl-tRNA synthetase. Chembiochem. 10: 2188-90. Pubmed
Xu LY, Link AJ. (2009) Stress responses to heterologous membrane protein expression in Escherichia coli. Biotechnol Lett. 31: 1775-82. Pubmed
Link AJ, Skretas G, Strauch EM, Chari NS, Georgiou G. (2008) Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli. Protein Sci. 17: 1857-63. Pubmed
Strable E, Prasuhn DE Jr, Udit AK,...Link AJ...Finn MG. (2008) Unnatural amino acid incorporation into virus-like particles. Bioconjug Chem. 19: 866-75. Pumed
Yoo TH, Link AJ, Tirrell DA. (2007) Evolution of a fluorinated green fluorescent protein. Proc Natl Acad Sci. 104: 13887-90. Pubmed
Link AJ, Jeong KJ, Georgiou G. (2007) Beyond toothpicks: new methods for isolating mutant bacteria. Nat Rev Microbiol. 5: 680-88. Pubmed
Link AJ, Vink MK, Tirrell DA. (2007) Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor. Nat Protoc. 2: 1884-87. Pubmed
Link AJ, Vink MK, Tirrell DA. (2007) Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer. Nat Protoc. 2: 1879-83. Pubmed
Link AJ, Georgiou G. (2007) Advances and challenges in membrane protein expression. AIChE J. 53: 752-56.
Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM. (2007) Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat Protoc. 2: 532-40. Pubmed
Link AJ, Vink MK, Agard NJ, Prescher JA, Bertozzi CR, Tirrell DA. (2006) Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc Natl Acad Sci. 103: 10180-85. Pubmed
Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM. (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc Natl Acad Sci. 103: 9482-87. Pubmed
Link AJ, Tirrell DA. (2005) Reassignment of sense codons in vivo. Methods. 36: 291-98. Pubmed
Link AJ, Vink MK, Tirrell DA. (2004) Presentation and detection of azide functionality in bacterial cell surface proteins. J Am Chem Soc. 126: 10598-602. Pubmed
Link AJ, Mock ML, Tirrell DA. (2003) Non-canonical amino acids in protein engineering. Curr Opin Biotechnol. 14: 603-09. Pubmed
Link AJ, Tirrell DA. (2003) Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J Am Chem Soc. 125: 11164-65. Pubmed
Bradley AZ, Link AJ, Biswas K,...Platz MS. (2000) Hydrogen abstraction on photolysis of a naphthocarborane. Tetrahedron Lett. 41: 8691-94.