title: Codon optimisation is key for pernisine expression in Escherichia coli.
abstract: BACKGROUND
  Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.
  
  
  RESULTS
  The challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised with a S355A mutation of a predicted active site (pernisineS355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni2+ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisinewt was not expressed in E. coli at detectable levels, while pernisineco and pernisineS355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisineco was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisineS355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.
  
  
  CONCLUSIONS
  These data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.
authors: Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors: Marko
authors: M
authors: Šnajder
authors: Centre of Excellence for Integrated Approaches in Chemistry and Biology (CipKeBiP), Ljubljana, Slovenia; Institute Jozef Stefan, Ljubljana, Slovenia.
authors: Marko
authors: M
authors: Mihelič
authors: Centre of Excellence for Integrated Approaches in Chemistry and Biology (CipKeBiP), Ljubljana, Slovenia; Institute Jozef Stefan, Ljubljana, Slovenia.
authors: Dušan
authors: D
authors: Turk
authors: Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; Centre of Excellence for Integrated Approaches in Chemistry and Biology (CipKeBiP), Ljubljana, Slovenia.
authors: Nataša Poklar
authors: NP
authors: Ulrih
mesh_terms: D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D057057:Serine Proteases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D003062:Codon; D057057:Serine Proteases