Novel tools for the study of protein-protein interactions in pluripotent cells

textUnnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described.Biomedical Engineerin

Photonic crystal Microarray Nanoplatform for High Throughput Detection of Biomolecules

ABSTRACT We present preliminary designs and experimental results for creating a microarray nanoplatform based on twodimensional photonic crystal devices in silicon. Multiple photonic crystal microcavities are coupled along the length of a single photonic crystal waveguide that undergo resonance wavelength shifts when an antibody-antibody binding event occurs in the immediate vicinity of the corresponding photonic crystal microcavity. The microarray nanoplatform enables high throughput measurements of multiple antibody-antibody interactions via a single optical waveguide transmission measurement

Transforming a Pair of Orthogonal tRNA-aminoacyl-tRNA Synthetase from Archaea to Function in Mammalian Cells

A previously engineered Methanocaldococcus jannaschii –tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting -tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells

Crucial optimization of translational components towards efficient incorporation of unnatural amino acids into proteins in mammalian cells.

The ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins is a powerful tool in protein engineering. While dozens of UAAs have been successfully introduced into proteins expressed by Escherichia coli cells, it has been much more challenging to create tRNA and tRNA-Synthetase pairs that enable UAAs incorporation, for use in mammalian systems. By altering the orthogonality properties of existing unnatural pairs, previously evolved pairs for use in E. coli could be used in mammalian cells. This would bypass the cumbersome step of having to evolve mutant synthetases and would allow for the rapid development of new mammalian pairs. A major limitation to the amount of UAA-containing proteins that can be expressed in the cell is the availability of UAA-charged orthogonal suppressor tRNA. By using a natural mammalian tRNA promoter, the amount of functional suppressor tRNA can be greatly increased. Furthermore, increasing recognition of the suppressor tRNA by the mutant synthetase will ultimately lead to the appearance of more UAA-charged tRNA

Alignment of the amino acid sequences of the CP1-transplanted mutants.

<p>Inserted CP1 sequences are shown in black. Six sequences used were from <i>E. coli</i> TyrRS, while two were taken from <i>T. thermophilus</i> TyrRS. Both bacterial TyrRSs recognize a G1:C72 containing tyrosyl-tRNA. Each CP1 swapped TyrRS was tested for the ability to charge 1bp-tRNA_CUA in HEK293T cells. (Bioworkbench, SDSC).</p

Assessing the orthogonality of various tRNA constructs in HEK293T cells.

<p>Full-length GFP was visualized by UV-light 72 hours after transfection. Expression of full-length GFP will only occur in the presence of amino acid charged suppressor tRNA. Since no exogenous aaRS was provided, full-length GFP expression implies that endogenous aaRS was able to recognize and charge the tRNA with an amino acid. Cells were transfected with the following plasmids: (A) p-EGFP-N1 (B) p-GFP_39TAG (C) p-GFP_39TAG and H1_wt-tRNA_CUA (D) p-GFP_39TAG and H1_1bp-tRNA_CUA (E) p-GFP_39TAG and H1_2bp-tRNA_CUA (F) p-GFP_39TAG and H1_3bp-tRNA_CUA.</p

Acceptor stem sequences of the various tRNA constructs.

<p>Each tRNA construct was cloned into a plasmid, downstream of the human H1 promoter. Wt-tRNA_CUA is identical to <i>M. jannaschii</i> tyrosyl-tRNA, except in the anticodon region, where it contains a C35 (pknotsRG, BiBiServ).</p

The orthogonality of the <i>M. jannaschii</i> TyrRS- pair was verified on western blots probed with anti-V5 antibodies.

<p>Expression of full-length foldon was monitored when various tRNAs were introduced into the HEK 293T cells. Note that the tRNA mutants used in these experiments were slightly different from those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011263#pone-0011263-g001" target="_blank">figure 1</a>.</p

Suppression of an amber codon inserted into the GFP-encoding gene.

<p>(A) Full-length GFP was expressed in HEK 293T cells only in the presence of a <i>M. jannaschii</i> TyrRS- pair designed to be orthogonal to mammalian cells. (B) Western blot analysis of full-length GFP probed with anti-His antibodies.</p