A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III.
The design of chimeric proteins is a major field of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors.
Trovato
M
M
Dipartimento di Genetica e Biologia Molecolare 'Charles Darwin', Università di Roma 'La Sapienza', Piazzale A. Moro 5, Rome, I-00185, Italy.
Maras
B
B
Polticelli
F
F
Costantino
P
P
Ascenzi
P
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001937:Brassica / Q000737:chemistry*; D002417:Cattle; D002846:Chromatography, Affinity; D004926:Escherichia coli; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin