Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium
Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (...
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MDPI AG
2023-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/14/11779 |
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| author | Tomas Jecmen Roman Tuzhilkin Miroslav Sulc |
| author_facet | Tomas Jecmen Roman Tuzhilkin Miroslav Sulc |
| author_sort | Tomas Jecmen |
| collection | DOAJ |
| description | Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic <i>E. coli</i> BL-21 and auxotrophic <i>E. coli</i> B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b<sub>5</sub> (b<sub>5</sub>M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic <i>E. coli</i>, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b<sub>5</sub>M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70–80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg. |
| first_indexed | 2024-03-11T00:59:37Z |
| format | Article |
| id | doaj.art-5a854b3576b04900a88198036cbe1009 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T00:59:37Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-5a854b3576b04900a88198036cbe10092023-11-18T19:44:53ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124141177910.3390/ijms241411779Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal MediumTomas Jecmen0Roman Tuzhilkin1Miroslav Sulc2Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague, Czech RepublicDepartment of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague, Czech RepublicDepartment of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 43 Prague, Czech RepublicResidue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic <i>E. coli</i> BL-21 and auxotrophic <i>E. coli</i> B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b<sub>5</sub> (b<sub>5</sub>M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic <i>E. coli</i>, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b<sub>5</sub>M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70–80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.https://www.mdpi.com/1422-0067/24/14/11779non-canonical amino-acid-containing proteinsbio-orthogonal amino acid global substitutionazidohomoalaninehomopropargylglycinephoto-methionine<i>E. coli</i> |
| spellingShingle | Tomas Jecmen Roman Tuzhilkin Miroslav Sulc Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium International Journal of Molecular Sciences non-canonical amino-acid-containing proteins bio-orthogonal amino acid global substitution azidohomoalanine homopropargylglycine photo-methionine <i>E. coli</i> |
| title | Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium |
| title_full | Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium |
| title_fullStr | Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium |
| title_full_unstemmed | Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium |
| title_short | Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic <i>E. coli</i> in Minimal Medium |
| title_sort | photo methionine azidohomoalanine and homopropargylglycine are incorporated into newly synthesized proteins at different rates and differentially affect the growth and protein expression levels of auxotrophic and prototrophic i e coli i in minimal medium |
| topic | non-canonical amino-acid-containing proteins bio-orthogonal amino acid global substitution azidohomoalanine homopropargylglycine photo-methionine <i>E. coli</i> |
| url | https://www.mdpi.com/1422-0067/24/14/11779 |
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