Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we...

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Autores principales:	Georgia-Persephoni Voulgaridou, Theodora Mantso, Katerina Chlichlia, Mihalis I Panayiotidis, Aglaia Pappa
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b15b5ecac5d04c2799a1b8302e5af4af
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spelling	oai:doaj.org-article:b15b5ecac5d04c2799a1b8302e5af4af2021-11-18T07:56:26ZEfficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.1932-620310.1371/journal.pone.0056582https://doaj.org/article/b15b5ecac5d04c2799a1b8302e5af4af2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23451057/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli's molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli's molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin.Georgia-Persephoni VoulgaridouTheodora MantsoKaterina ChlichliaMihalis I PanayiotidisAglaia PappaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 2, p e56582 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Georgia-Persephoni Voulgaridou Theodora Mantso Katerina Chlichlia Mihalis I Panayiotidis Aglaia Pappa Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
description	Aldehyde dehydrogenase 3A1 (ALDH3A1) is a recently characterized corneal crystallin with its exact functions still being unclear. Expressing recombinant human ALDH3A1 has been difficult in Escherichia coli (E. coli) because of low solubility, yield and insufficient purity issues. In this report, we compared different E. coli expression strategies (namely the maltose binding protein; MBP- and the 6-his-tagged expression systems) under conditions of auto-induction and co-expression with E. coli's molecular chaperones where appropriate. Thus, we aimed to screen the efficiency of these expression strategies in order to improve solubility of recombinant ALDH3A1 when expressed in E. coli. We showed that the MBP- tagged expression in combination with lower-temperature culture conditions resulted in active soluble recombinant ALDH3A1. Expression of the fused 6-his tagged-ALDH3A1 protein resulted in poor solubility and neither lowering temperature culture conditions nor the auto-induction strategy improved its solubility. Furthermore, higher yield of soluble, active native form of 6-his tagged-ALDH3A1 was facilitated through co-expression of the two groups of E. coli's molecular chaperones, GroES/GroEL and DnaK/DnaJ/GrpE. Convenient one step immobilized affinity chromatography methods were utilized to purify the fused ALDH3A1 hybrids. Both fusion proteins retained their biological activity and could be used directly without removing the fusion tags. Taken together, our results provide a rational option for producing sufficient amounts of soluble and active recombinant ALDH3A1 using the E. coli expression system for conducting functional studies towards elucidating the biological role(s) of this interesting corneal crystallin.
format	article
author	Georgia-Persephoni Voulgaridou Theodora Mantso Katerina Chlichlia Mihalis I Panayiotidis Aglaia Pappa
author_facet	Georgia-Persephoni Voulgaridou Theodora Mantso Katerina Chlichlia Mihalis I Panayiotidis Aglaia Pappa
author_sort	Georgia-Persephoni Voulgaridou
title	Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
title_short	Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
title_full	Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
title_fullStr	Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
title_full_unstemmed	Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
title_sort	efficient e. coli expression strategies for production of soluble human crystallin aldh3a1.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/b15b5ecac5d04c2799a1b8302e5af4af
work_keys_str_mv	AT georgiapersephonivoulgaridou efficientecoliexpressionstrategiesforproductionofsolublehumancrystallinaldh3a1 AT theodoramantso efficientecoliexpressionstrategiesforproductionofsolublehumancrystallinaldh3a1 AT katerinachlichlia efficientecoliexpressionstrategiesforproductionofsolublehumancrystallinaldh3a1 AT mihalisipanayiotidis efficientecoliexpressionstrategiesforproductionofsolublehumancrystallinaldh3a1 AT aglaiapappa efficientecoliexpressionstrategiesforproductionofsolublehumancrystallinaldh3a1
_version_	1718422750715969536

Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.

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