Selenocysteine in proteins : Properties and biotechnological use

Abstract: Selenocysteine (Sec), the 21st amino acid, exists in all kingdoms of life and has unique biochemical properties, such as high electrophilicity and low pKa. The highly increased reactivity of selenoenzymes compared to their sulfur-containing cysteine-dependent homologs is generally regarded as the evolutionary reason for having selenoproteins. A Sec residue of a selenoprotein is co-translationally incorporated at a predefined UGA-codon. This is re-coded from a termination codon to Sec-encoding by species-specific translation mechanisms dependent on structural features of the mRNA. As a consequence, selenoproteins have generally been excluded from conventional heterologous recombinant expression in bacteria. However, our group has been able to by-pass this species-barrier and successfully expressed the mammalian selenoprotein thioredoxin reductase (TrxR) in E. coli. The technique for expressing recombinant selenoproteins in E. coli involves tailoring of genes to become compatible with the bacterial selenoprotein synthesis machinery. In this thesis, this methodology has been used for studying the properties of Sec in proteins. The possibility of using Sec for a wide range of biotechnological applications has also been explored and demonstrated. I) TrxR of D. melanogaster naturally contains Cys instead of Sec in the active site, but surprisingly has nearly the same catalytic activity as the mammalian counterpart. We found that the catalytic rate of the insect enzyme is highly dependent on two serine residues, which somehow activate the redox active Cys to act more like a Sec moiety. Our results suggest that selenocysteine is not necessary for a high catalytic efficiency per se but gives an advantage of a broader range of substrates and a wider range of environmental conditions within which the catalytic efficiency can be maintained. II) In mammalian TrxR the Sec moiety is the penultimate residue, which, due to the constraints of selenoprotein mRNA structural features, facilitated its expression as a recombinant protein in E. coli. Producing selenoproteins with a Sec residue internally positioned is more problematic. Despite the technical difficulty, a Sec-substituted GST could nonetheless be produced at a yield of 2,9 mg/l bacterial culture, showing a promising potential for the technique to be applied in recombinant production also of certain proteins with internal Sec residues. III) The C-terminal motif of mammalian TrxR, -Gly-Cys-Sec-Gly, was introduced as a fusion motif for recombinant proteins produced in E. coli, named a Sel-tag. Human Vasoactive Intestinal Peptide (VIP) and the dust mite allergen Der p 2, served the basis for development and evaluation of Sel-tag based techniques. We found that the Sel-tag could be used as a protein tag for purification of the recombinant protein, the basis for selenolate-targeted labeling with fluorescent compounds, or radiolabeling with either gammaemitting 75Se or short-lived positron-emitters such as 11C. IV) We have demonstrated an in vivo application of the Sel-tag. The dust mite allergen Der p 2 was thus labeled with 75Se and used for tracking in vivo allergen uptake in a mouse model for mite allergy. The fate of the labeled allergen was followed after intratracheal administration at the whole body level as well as on the protein level by whole body autoradiography and tissue extractions. We found that the inflammatory state of the lung upon allergen challenge influenced the clearance of Der p 2. Thus an allergic response to the allergen may lead to prolonged retention of Der p 2 in the lung.