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A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies

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Standard

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. / Linova, Marina Y.; Risør, Michael W.; Jørgensen, Sanne E.; Mansour, Zohra; Kaya, Jacob; Sigurdarson, Jens J.; Enghild, Jan J.; Karring, Henrik.

In: Protein Expression and Purification, Vol. 166, 105507, 01.02.2020.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Harvard

Linova, MY, Risør, MW, Jørgensen, SE, Mansour, Z, Kaya, J, Sigurdarson, JJ, Enghild, JJ & Karring, H 2020, 'A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies', Protein Expression and Purification, vol. 166, 105507. https://doi.org/10.1016/j.pep.2019.105507

APA

Linova, M. Y., Risør, M. W., Jørgensen, S. E., Mansour, Z., Kaya, J., Sigurdarson, J. J., ... Karring, H. (2020). A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. Protein Expression and Purification, 166, [105507]. https://doi.org/10.1016/j.pep.2019.105507

CBE

Linova MY, Risør MW, Jørgensen SE, Mansour Z, Kaya J, Sigurdarson JJ, Enghild JJ, Karring H. 2020. A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. Protein Expression and Purification. 166. https://doi.org/10.1016/j.pep.2019.105507

MLA

Vancouver

Linova MY, Risør MW, Jørgensen SE, Mansour Z, Kaya J, Sigurdarson JJ et al. A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. Protein Expression and Purification. 2020 Feb 1;166. 105507. https://doi.org/10.1016/j.pep.2019.105507

Author

Linova, Marina Y. ; Risør, Michael W. ; Jørgensen, Sanne E. ; Mansour, Zohra ; Kaya, Jacob ; Sigurdarson, Jens J. ; Enghild, Jan J. ; Karring, Henrik. / A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. In: Protein Expression and Purification. 2020 ; Vol. 166.

Bibtex

@article{17d96667d31d4777a6e95a1eaf7c6bdd,
title = "A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies",
abstract = "The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403–621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416–621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying L-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M L-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.",
keywords = "Inclusion bodies, L-arginine, Refolding, Small ubiquitin-like modifier (SUMO), SUMO-Specific protease, Ubiquitin-like protease (Ulp)",
author = "Linova, {Marina Y.} and Ris{\o}r, {Michael W.} and J{\o}rgensen, {Sanne E.} and Zohra Mansour and Jacob Kaya and Sigurdarson, {Jens J.} and Enghild, {Jan J.} and Henrik Karring",
year = "2020",
month = "2",
day = "1",
doi = "10.1016/j.pep.2019.105507",
language = "English",
volume = "166",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",

}

RIS

TY - JOUR

T1 - A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies

AU - Linova, Marina Y.

AU - Risør, Michael W.

AU - Jørgensen, Sanne E.

AU - Mansour, Zohra

AU - Kaya, Jacob

AU - Sigurdarson, Jens J.

AU - Enghild, Jan J.

AU - Karring, Henrik

PY - 2020/2/1

Y1 - 2020/2/1

N2 - The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403–621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416–621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying L-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M L-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

AB - The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403–621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416–621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying L-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M L-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

KW - Inclusion bodies

KW - L-arginine

KW - Refolding

KW - Small ubiquitin-like modifier (SUMO)

KW - SUMO-Specific protease

KW - Ubiquitin-like protease (Ulp)

UR - http://www.scopus.com/inward/record.url?scp=85072995513&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2019.105507

DO - 10.1016/j.pep.2019.105507

M3 - Journal article

C2 - 31586598

AN - SCOPUS:85072995513

VL - 166

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

M1 - 105507

ER -