title: Decreasing translation error rate in Escherichia coli increases protein function.
abstract: BACKGROUND
  Over-expressed native or recombinant proteins are commonly used for industrial and pharmaceutical purposes, as well as for research. Proteins of interest need to be purified in sufficient quantity, quality and specific activity to justify their commercial price and eventual medical use. Proteome quality was previously positively correlated with ribosomal fidelity, but not on a single protein level. Here, we show that decreasing translational error rate increases the activity of single proteins. In order to decrease the amount of enzyme needed for catalysis, we propose an expression system bearing rpsL141 mutation, which confers high ribosomal fidelity. Using alpha-glucosidase (exo-alpha-1,4-glucosidase) and beta-glucanase (beta-D-glucanase) as examples, we show that proteins purified from Escherichia coli bearing rpsL141 mutation have superior activity compared to those purified from wild type E. coli, as well as some commercially available industrial enzymes.
  
  
  RESULTS
  Our results indicate that both alpha-glucosidase and beta-glucanase isolated from E. coli bearing rpsL141 mutation have increased activity compared to those isolated from wild type E. coli. Alpha-glucosidase from rpsL141 background has a higher activity than the purchased enzymes, while beta-glucanase from the same background has a higher activity compared to the beta-glucanase purchased from Sigma, but not compared to the one purchased from Megazyme.
  
  
  CONCLUSIONS
  Reduction of the error rate in protein biosynthesis via ribosomal rpsL141 mutation results in superior functionality of single proteins. We conclude that this is a viable system for expressing proteins with higher activity and that it can be easily scaled up and combined with other expression systems to meet the industrial needs.
authors.affiliation: Mediterranean Institute for Life Sciences (MedILS), Mestrovicevo setaliste 45, 21000, Split, Croatia.
authors.forename: Marina
authors.initials: M
authors.lastname: Musa
authors.affiliation: Mediterranean Institute for Life Sciences (MedILS), Mestrovicevo setaliste 45, 21000, Split, Croatia.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Radman
authors.affiliation: Mediterranean Institute for Life Sciences (MedILS), Mestrovicevo setaliste 45, 21000, Split, Croatia. anita.krisko@medils.hr.
authors.forename: Anita
authors.initials: A
authors.lastname: Krisko
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D050050:Protein Carbonylation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C506766:RpsL protein, E coli; D005959:Glucosidases
keywords: Protein activity; Protein expression systems; rpsL141