Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translati...
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Autor*in:	Puertas, Juan-Miguel [verfasserIn] Betton, Jean-Michel

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2009

Schlagwörter:	Signal Sequence Bias Codon Usage Codon Optimization Signal Recognition Particle High Cell Density Culture

Anmerkung:	© Puertas and Betton; licensee BioMed Central Ltd. 2009

Übergeordnetes Werk:	Enthalten in: Microbial cell factories - London : Biomed Central, 2002, 8(2009), 1 vom: 29. Okt.
Übergeordnetes Werk:	volume:8 ; year:2009 ; number:1 ; day:29 ; month:10

Links:	Volltext

DOI / URN:	10.1186/1475-2859-8-57

Katalog-ID:	SPR028557417

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520			\|a Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli.
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allfields	10.1186/1475-2859-8-57 doi (DE-627)SPR028557417 (SPR)1475-2859-8-57-e DE-627 ger DE-627 rakwb eng Puertas, Juan-Miguel verfasserin aut Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Puertas and Betton; licensee BioMed Central Ltd. 2009 Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213 Betton, Jean-Michel aut Enthalten in Microbial cell factories London : Biomed Central, 2002 8(2009), 1 vom: 29. Okt. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:8 year:2009 number:1 day:29 month:10 https://dx.doi.org/10.1186/1475-2859-8-57 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2009 1 29 10
spelling	10.1186/1475-2859-8-57 doi (DE-627)SPR028557417 (SPR)1475-2859-8-57-e DE-627 ger DE-627 rakwb eng Puertas, Juan-Miguel verfasserin aut Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Puertas and Betton; licensee BioMed Central Ltd. 2009 Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213 Betton, Jean-Michel aut Enthalten in Microbial cell factories London : Biomed Central, 2002 8(2009), 1 vom: 29. Okt. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:8 year:2009 number:1 day:29 month:10 https://dx.doi.org/10.1186/1475-2859-8-57 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2009 1 29 10
allfields_unstemmed	10.1186/1475-2859-8-57 doi (DE-627)SPR028557417 (SPR)1475-2859-8-57-e DE-627 ger DE-627 rakwb eng Puertas, Juan-Miguel verfasserin aut Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Puertas and Betton; licensee BioMed Central Ltd. 2009 Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213 Betton, Jean-Michel aut Enthalten in Microbial cell factories London : Biomed Central, 2002 8(2009), 1 vom: 29. Okt. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:8 year:2009 number:1 day:29 month:10 https://dx.doi.org/10.1186/1475-2859-8-57 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2009 1 29 10
allfieldsGer	10.1186/1475-2859-8-57 doi (DE-627)SPR028557417 (SPR)1475-2859-8-57-e DE-627 ger DE-627 rakwb eng Puertas, Juan-Miguel verfasserin aut Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Puertas and Betton; licensee BioMed Central Ltd. 2009 Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213 Betton, Jean-Michel aut Enthalten in Microbial cell factories London : Biomed Central, 2002 8(2009), 1 vom: 29. Okt. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:8 year:2009 number:1 day:29 month:10 https://dx.doi.org/10.1186/1475-2859-8-57 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2009 1 29 10
allfieldsSound	10.1186/1475-2859-8-57 doi (DE-627)SPR028557417 (SPR)1475-2859-8-57-e DE-627 ger DE-627 rakwb eng Puertas, Juan-Miguel verfasserin aut Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli 2009 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Puertas and Betton; licensee BioMed Central Ltd. 2009 Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213 Betton, Jean-Michel aut Enthalten in Microbial cell factories London : Biomed Central, 2002 8(2009), 1 vom: 29. Okt. (DE-627)355987651 (DE-600)2091377-1 1475-2859 nnns volume:8 year:2009 number:1 day:29 month:10 https://dx.doi.org/10.1186/1475-2859-8-57 kostenfrei Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2009 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2055 GBV_ILN_2111 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 8 2009 1 29 10
language	English
source	Enthalten in Microbial cell factories 8(2009), 1 vom: 29. Okt. volume:8 year:2009 number:1 day:29 month:10
sourceStr	Enthalten in Microbial cell factories 8(2009), 1 vom: 29. Okt. volume:8 year:2009 number:1 day:29 month:10
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Signal Sequence Bias Codon Usage Codon Optimization Signal Recognition Particle High Cell Density Culture
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container_title	Microbial cell factories
authorswithroles_txt_mv	Puertas, Juan-Miguel @@aut@@ Betton, Jean-Michel @@aut@@
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author	Puertas, Juan-Miguel
spellingShingle	Puertas, Juan-Miguel misc Signal Sequence misc Bias Codon Usage misc Codon Optimization misc Signal Recognition Particle misc High Cell Density Culture Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
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topic_title	Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli Signal Sequence (dpeaa)DE-He213 Bias Codon Usage (dpeaa)DE-He213 Codon Optimization (dpeaa)DE-He213 Signal Recognition Particle (dpeaa)DE-He213 High Cell Density Culture (dpeaa)DE-He213
topic	misc Signal Sequence misc Bias Codon Usage misc Codon Optimization misc Signal Recognition Particle misc High Cell Density Culture
topic_unstemmed	misc Signal Sequence misc Bias Codon Usage misc Codon Optimization misc Signal Recognition Particle misc High Cell Density Culture
topic_browse	misc Signal Sequence misc Bias Codon Usage misc Codon Optimization misc Signal Recognition Particle misc High Cell Density Culture
format_facet	Elektronische Aufsätze Aufsätze Elektronische Ressource
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title	Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
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title_full	Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
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author_browse	Puertas, Juan-Miguel Betton, Jean-Michel
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title_sort	engineering an efficient secretion of leech carboxypeptidase inhibitor in escherichia coli
title_auth	Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
abstract	Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. © Puertas and Betton; licensee BioMed Central Ltd. 2009
abstractGer	Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. © Puertas and Betton; licensee BioMed Central Ltd. 2009
abstract_unstemmed	Background Despite advances in expression technologies, the efficient production of heterologous secreted proteins in Escherichia coli remains a challenge. One frequent limitation relies on their inability to be exported to the E. coli periplasm. However, recent studies have suggested that translational kinetics and signal sequences act in concert to modulate the export process. Results In order to produce leech carboxypeptidase inhibitor (LCI) in the bacterial periplasm, we compared expression of the natural and optimized gene sequences, and evaluated export efficiency of LCI fused to different signal sequences. The best combination of these factors acting on translation and export was obtained when the signal sequence of DsbA was fused to an E. coli codon-optimized mature LCI sequence. When tested in high cell density cultures, the protein was primarily found in the growth medium. Under these conditions, the engineered expression system yields over 470 mg.$ l^{-1} $ of purified active LCI. Conclusion These results support the hypothesis that heterologous secreted proteins require proper coupling between translation and translocation for optimal high-level production in E. coli. © Puertas and Betton; licensee BioMed Central Ltd. 2009
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title_short	Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli
url	https://dx.doi.org/10.1186/1475-2859-8-57
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Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli

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