title: Efficient E. coli expression strategies for production of soluble human crystallin ALDH3A1.
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.
authors: Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, Alexandroupolis, Greece.
authors: Georgia-Persephoni
authors: GP
authors: Voulgaridou
authors: Theodora
authors: T
authors: Mantso
authors: Katerina
authors: K
authors: Chlichlia
authors: Mihalis I
authors: MI
authors: Panayiotidis
authors: Aglaia
authors: A
authors: Pappa
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008957:Models, Genetic; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
chemical_list: D018832:Molecular Chaperones; D011994:Recombinant Proteins; C480021:ALDH3A1 protein, human; D000444:Aldehyde Dehydrogenase