Genetic encoding of sulfotyrosine (sTyr) in mammalian cells

Xinyuan He Author

04/05/2021 Added

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

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[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.

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Genetic encoding of sulfotyrosine (sTyr) in mammalian cells

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