Expression of Bv8 in Pichia pastoris to identify structural features for receptor binding.
Bv8 is an amphibian peptide belonging to the widely distributed AVIT protein family. The mammalian orthologues of Bv8 were named prokineticin 1 and prokineticin 2. Two G-protein-coupled receptors for Bv8-prokineticins have been identified. The biological activities of Bv8/PK proteins range from angiogenesis and involvement in reproduction and cancer, to neuronal survival and neurogenesis, hypothalamic hormone secretion, circadian rhythm control and immunomodulatory processes. Identifying the structural determinants required for receptor binding of Bv8-PKs is mandatory for the design of PKR antagonists, which may be useful in the treatment and prevention of various disease states. Here we describe a procedure for the production in Pichia pastoris of Bv8 and 3 mutants: W24A-Bv8, in which the tryptophan in position 24 is substituted by alanine, the double mutant M1-W24A-Bv8, that contains an additional methionine at the N-terminus and Bv8-TyrTyr that includes two additional tyrosines at the C-terminus. The results evidence a relevant role of tryptophan 24 in Bv8-PKRs interaction.
Miele
Rossella
R
Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche A. Rossi Fanelli and CNR Istituto di Biologia e Patologia Molecolari, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy. rossella.miele@uniroma1.it
Lattanzi
Roberta
R
Bonaccorsi di Patti
Maria Carmela
MC
Paiardini
Alessandro
A
Negri
Lucia
L
Barra
Donatella
D
D029845:Amphibian Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001001:Anura / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009479:Neuropeptides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan
D029845:Amphibian Proteins; C121344:Bv8 protein, Bombina variegata; D009479:Neuropeptides; D011994:Recombinant Proteins; D014364:Tryptophan