Assessment of production conditions for efficient use of Escherichia coli in high-yield heterologous recombinant selenoprotein synthesis.

The production of heterologous selenoproteins in Escherichia coli necessitates the design of a secondary structure in the mRNA forming a selenocysteine insertion sequence (SECIS) element compatible with SelB, the elongation factor for selenocysteine insertion at a predefined UGA codon. SelB competes with release factor 2 (RF2) catalyzing translational termination at UGA. Stoichiometry between mRNA, the SelB elongation factor, and RF2 is thereby important, whereas other expression conditions affecting the yield of recombinant selenoproteins have been poorly assessed. Here we expressed the rat selenoprotein thioredoxin reductase, with titrated levels of the selenoprotein mRNA under diverse growth conditions, with or without cotransformation of the accessory bacterial selA, selB, and selC genes. Titration of the selenoprotein mRNA with a pBAD promoter was performed in both TOP10 and BW27783 cells, which unexpectedly could not improve yield or specific activity compared to that achieved in our prior studies. Guided by principal component analysis, we instead discovered that the most efficient bacterial selenoprotein production conditions were obtained with the high-transcription T7lac-driven pET vector system in presence of the selA, selB, and selC genes, with induction of production at late exponential phase. About 40 mg of rat thioredoxin reductase with 50% selenocysteine content could thereby be produced per liter bacterial culture. These findings clearly illustrate the ability of E. coli to upregulate the selenocysteine incorporation machinery on demand and that this is furthermore strongly augmented in late exponential phase. This study also demonstrates that E. coli can indeed be utilized as cell factories for highly efficient production of heterologous selenoproteins such as rat thioredoxin reductase.

A structure-activity study of nociceptin-(1-13)-peptide amide. Synthesis of analogues substituted in positions 0, 1, 3, 4 and 10.

A series of analogues of nociceptin, Noc(1-13)NH(2) (an agonist at the ORL1 receptor) was synthesized with following modifications: (1) N-terminal extension with Arg(0); (2) replacement of Gly(3) by basic or polar amino acids-Arg, Asn, Lys(For) or deletion; (3) exchange of Phe(1) or Phe(4) by Phe(NO(2)); (4) substitution of Ser(10) with D-Ser, Pro, D-Pro. The analogs were synthesized by solid-phase methodology using Fmoc-amino acid pentafluorophenyl esters. The affinity for the ORL1 and for the kappa, micro and delta-opioid receptors was investigated by radioligand binding assay and bioactivity by a mouse vas deferens (MVD) assay. The addition of the amino acid residue Arg to the N-terminal enhances the opioid receptor affinity of Noc(1-13)NH(2) while retaining ORL1 receptor affinity at a moderate level. The replacement of Gly in position 3 by the basic or polar amino acids-Arg, Asn, Lys(For) or its deletion led to inactive analogues. The replacement of Ser in position 10 by its D-isomer, Pro and D-Pro resulted in a series of analogues with the following order of activity: Ser(10)>D-Ser(10)>Pro(10)>D-Pro(10). In [D-Ser(10)]Noc(1-13)NH(2), introduction of an additional Phe(NO(2))(4) led to a >60-fold increase of ORL1 affinity, completely attenuating the loss of affinity brought about by Ser(10). In other analogues, introduction of Phe(NO(2))(4) did not change the magnitude of ORL1 binding significantly. Generally, while modifications in position 3 frequently led to a loss of most or all bioactivity, modifications in position 0 (Arg(0)) or 4 (Phe(NO(2))(4)) and 10 (D-Ser(10), Pro(10)) are tolerated.