Genetic encoding of sulfotyrosine (sTyr) in mammalian cells

Xinyuan He Author

04/05/2021 Added

22 Plays

Description

We engineered an E. coli tyrosyl-tRNA synthetase that can translationally incorporate sTyr in response to UAG codons in yeast and mammalian cells.

Searchable Transcript

Search:

[00:00:01.710]Hi, welcome to my presentation. My name is Xinyuan He.
[00:00:05.340]I'm currently a Ph.D. student from Biomedical engineering program.
[00:00:10.410]My advisor is Dr. Wei Niu.
[00:00:15.210]Here, I would like to share our recent work on genetic encoding of sulfotyrosine
[00:00:19.140]in mammalian cells. So, first of all,
[00:00:23.580]this is a brief introduction. Protein tyrosine o-sulfation
[00:00:27.780]is a post-translational modification that occurs,
[00:00:32.520]especially in plants and animals,
[00:00:35.310]where a sulfate group is added to a tyrosine residue of a
[00:00:40.050]protein.
[00:00:41.430]The introduction of negatively charged sulfate groups plays important roles
[00:00:46.380]in various cellular processes,
[00:00:49.050]including cell adhesion, hormone activities, and the immune
[00:00:53.340]responses. However, the physiological roles of protein
[00:00:57.990]tyrosine sulfation are not well understood.
[00:01:03.720]The key to study protein
[00:01:05.550]tyrosine sulfation is the ability to synthesize site-specifically
[00:01:09.930]sulfated proteins. Here we engineered an
[00:01:13.920]E. coli tyrosyl-tRNA synthetase that can translationally incorporate a
[00:01:18.540]sulfotyrosine in response to the amber codon in yeast
[00:01:23.280]and in mammalian cells.
[00:01:26.190]The method was developed in 2003. To evolve a tRNA synthetase
[00:01:31.170]that can recognize sTyr,
[00:01:36.180]S. cerevisiae Mav203
[00:01:36.910]was used as a selection host.
[00:01:41.320]In Mav203,
[00:01:42.150]URA3 gene is under the control of the transcriptional
[00:01:46.410]activator GAL4,
[00:01:47.760]which contains two amber codons.
[00:01:52.310]In the positive selection, yeast cells were cultured in the presence of
[00:01:57.000]sTyr
[00:01:58.680]Effective amber suppression by mutants that can charge sTyr
[00:02:03.660]enables the growth of Mav203 without uracil.
[00:02:07.890]In the negative selection, Yeast cells were grown
[00:02:12.720]in 5-FOA.
[00:02:17.520]Undesirable aminoacylation with any of
[00:02:21.180]the 20 natural amino acids led to the conversion of 5-FOA into
[00:02:26.020]toxic compound and caused cell death. Overall,
[00:02:30.720]survived candidates are supposed to specifically aminoacylate
[00:02:34.860]sTyr to its orthogonal tRNA.
[00:02:40.890]Here is the result. After selection,
[00:02:44.220]two mutants were identified. And the sequences show
[00:02:48.930]great consensus.
[00:02:51.420]The two mutants were first evaluated in Mav203
[00:02:56.340]using yeGFP as a reporter gene. Mutant c2
[00:03:01.090]showed 3-fold GFP expression with 1 mM and
[00:03:05.890]5 mM sTyr,
[00:03:08.020]While mutant c1 can only show similar activity in the presence of
[00:03:12.850]5 mM sTyr.
[00:03:15.310]This suggests that both C1 and C2 were able to
[00:03:19.990]aminoacylate tRNA with sTyr in yeast,
[00:03:24.100]and the C2 seemed to be more efficient.
[00:03:28.600]We also perform in vitro aminoacylation assay to
[00:03:33.100]confirm the enzymatic activity to sTyr. However,
[00:03:37.810]the C1 showed significant activity to both Tyr and sTyr,
[00:03:42.520]and C2 showed more
[00:03:46.810]activity to sTyr.
[00:03:49.870]Therefore, C2 was chosen in the following study.
[00:03:56.970]So next, we tried the
[00:04:01.800]tRNA synthetase in mammalian cells. eGFP gene was used
[00:04:06.720]as a reporter gene. So from the confocal image,
[00:04:10.470]although detectable background was observed without sTyr,
[00:04:15.930]significantly higher eGFP expression level was detected in the presence
[00:04:20.880]of sTyr. By using flow cytometry,
[00:04:26.130]The
[00:04:26.640]suppression efficiency was determined as 68%.
[00:04:32.190]So this is significantly higher than the well established
[00:04:37.350]AzF tRNA synthetase,
[00:04:40.230]which can efficiently incorporate azido-phenylalanine
[00:04:44.940]in mammalian cells.
[00:04:47.790]The fidelity of the evolved sTyr tRNA synthetase was
[00:04:51.970]quantified by mass spectrometry.
[00:04:55.050]The purified protein was analyzed by a time of
[00:05:00.000]flight mass spectrometer. From the result,
[00:05:04.740]No significant misincorporation of Tyr
[00:05:08.760]was observed when
[00:05:11.730]sTyr was added in the cell culture.
[00:05:16.950]So in summary,
[00:05:18.420]we developed an approach to site- specifically incorporate sTyr
[00:05:23.280]in proteins of interest in eukaryotic cells,
[00:05:27.750]with very high efficiency and fedility.
[00:05:31.470]This will facilitate the study of protein tyrosine sulfation
[00:05:35.890]-associated signaling pathways. In addition,
[00:05:41.880]the genetic incorporation of sTyr
[00:05:46.860]is expected to enable a high production of therapeutic proteins
[00:05:51.960]containing sTyr.
[00:05:56.070]We would like to thank Terri for
[00:05:58.100]Confocal imaging and Dirk Anderson for flow cytometry
[00:06:02.300]analysis.
[00:06:03.650]And this work was supported by startup fund
[00:06:07.340]from UNL and the fundings from NSF and NIH.
[00:06:12.770]With that, I would like to thank you again for coming to my presentation.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Genetic encoding of sulfotyrosine (sTyr) in mammalian cells

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments