Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins
Abstract Background Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a “sandwiched” SUMO-peptide-intein (SPI)...
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| Format: | Article |
| Language: | English |
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BMC
2023-04-01
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| Series: | BMC Biotechnology |
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| Online Access: | https://doi.org/10.1186/s12896-023-00779-5 |
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| author | Tess Lamer John C. Vederas |
| author_facet | Tess Lamer John C. Vederas |
| author_sort | Tess Lamer |
| collection | DOAJ |
| description | Abstract Background Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a “sandwiched” SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create “sandwiched” fusion proteins of the form: His6-SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein “sandwich” approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-“sandwiched”) fusion protein system. Results In this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His6-SUMO-peptide-intein-CBD-His6. The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol. Conclusions This modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue. |
| first_indexed | 2024-04-09T18:54:11Z |
| format | Article |
| id | doaj.art-bdc4ab2fb6994f3f830dfdb61bda1536 |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-09T18:54:11Z |
| publishDate | 2023-04-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-bdc4ab2fb6994f3f830dfdb61bda15362023-04-09T11:20:11ZengBMCBMC Biotechnology1472-67502023-04-0123111110.1186/s12896-023-00779-5Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteinsTess Lamer0John C. Vederas1Department of Chemistry, University of AlbertaDepartment of Chemistry, University of AlbertaAbstract Background Some peptides are targets for degradation when heterologously expressed as fusion proteins in E. coli, which can limit yields after isolation and purification. We recently reported that peptide degradation may be prevented by production of a “sandwiched” SUMO-peptide-intein (SPI) fusion protein, which protects the target peptide sequence from truncation and improves yield. This initial system required cloning with two commercially available vectors. It used an N-terminal polyhistidine tagged small ubiquitin-like modifier (SUMO) protein and a C-terminal engineered Mycobacterium xenopii DNA Gyrase A intein with an inserted chitin binding domain (CBD) to create “sandwiched” fusion proteins of the form: His6-SUMO-peptide-intein-CBD. However, the major drawback of this previously reported fusion protein “sandwich” approach is the increased time and number of steps required to complete the cloning and isolation procedures, relative to the simple procedures to produce recombinant peptides in E. coli from a single (non-“sandwiched”) fusion protein system. Results In this work we generate the plasmid pSPIH6, which improves upon the previous system by encoding both the SUMO and intein proteins and allows facile construction of a SPI protein in a single cloning step. Additionally, the Mxe GyrA intein encoded in pSPIH6 contains a C-terminal polyhistidine tag, resulting in SPI fusion proteins of the form: His6-SUMO-peptide-intein-CBD-His6. The dual polyhistidine tags greatly simplify isolation procedures compared to the original SPI system, which we have here demonstrated with two linear bacteriocin peptides: leucocin A and lactococcin A. The yields obtained for both peptides after purification were also improved compared to the previous SPI system as a result of this streamlined protocol. Conclusions This modified SPI system and its simplified cloning and purification procedures described here may be generally useful as a heterologous E. coli expression system to obtain pure peptides in high yield, especially when degradation of the target peptide is an issue.https://doi.org/10.1186/s12896-023-00779-5PeptideBacteriocinHeterologous expressionSmall ubiquitin-like modifier (SUMO)InteinProtein purification |
| spellingShingle | Tess Lamer John C. Vederas Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins BMC Biotechnology Peptide Bacteriocin Heterologous expression Small ubiquitin-like modifier (SUMO) Intein Protein purification |
| title | Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins |
| title_full | Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins |
| title_fullStr | Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins |
| title_full_unstemmed | Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins |
| title_short | Simplified cloning and isolation of peptides from “sandwiched” SUMO-peptide-intein fusion proteins |
| title_sort | simplified cloning and isolation of peptides from sandwiched sumo peptide intein fusion proteins |
| topic | Peptide Bacteriocin Heterologous expression Small ubiquitin-like modifier (SUMO) Intein Protein purification |
| url | https://doi.org/10.1186/s12896-023-00779-5 |
| work_keys_str_mv | AT tesslamer simplifiedcloningandisolationofpeptidesfromsandwichedsumopeptideinteinfusionproteins AT johncvederas simplifiedcloningandisolationofpeptidesfromsandwichedsumopeptideinteinfusionproteins |