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

Georgia-Persephoni Voulgaridou, Theodora Mantso, Katerina Chlichlia, Mihalis I Panayiotidis, Aglaia Pappa

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    Abstract

    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.
    Original languageEnglish
    Pages (from-to)e56582
    JournalPLoS ONE
    Volume8
    Issue number2
    Early online date22 Feb 2013
    DOIs
    Publication statusPublished - 2013

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    Voulgaridou, G-P., Mantso, T., Chlichlia, K., Panayiotidis, M. I., & Pappa, A. (2013). Efficient E.coli expression strategies for production of soluble human crystallin ALDH3A1. PLoS ONE, 8(2), e56582. https://doi.org/10.1371/journal.pone.0056582