title: Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration.
abstract: BACKGROUND
  Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli) remains challenging sometimes.
  
  
  RESULTS
  An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH) was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The kcat of this enzyme was 325 s-1 and the Km values for 6-phosphogluconate, NADP+, and NAD+ were 11, 10 and 380 muM, respectively, at 80 degrees C. This enzyme had half-life times of 48 and 140 h at 90 and 80 degrees C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate.
  
  
  CONCLUSIONS
  We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli.
authors.affiliation: Biological Systems Engineering Department, 210-A Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, Virgina 24061, USA. ypzhang@vt.edu.
authors.forename: Yiran
authors.initials: Y
authors.lastname: Wang
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang