High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.
We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.
Bianchi
E
E
Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia, Italy.
Venturini
S
S
Pessi
A
A
Tramontano
A
A
Sollazzo
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA; D004926:Escherichia coli; D005813:Genes, Synthetic; D007136:Immunoglobulins; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; D008670:Metals; C081296:Minibody (WT); D011994:Recombinant Proteins; D004247:DNA