title: Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems.
abstract: Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Dadashipour
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Fukuta
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007891:Leishmania / Q000378:metabolism; D002365:Manihot / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D000446:Aldehyde-Lyases