Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops.
Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillussubtilis expression system. The low purification yield is primarily due to a higher fraction of molecules with incorrect disulfide bond configurations after production and also after disulfide bond shuffling induced by 2-mercaptoethanol. To improve production yields, site-saturation libraries were generated at 39 out of the 66 amino acid residues of BBI-AV. Initial screens were designed to select for variants with higher trypsin inhibitory activities than the parent after treatment with a reducing agent. Secondary screens were developed to select for variants with the highest purification yields, and to also eliminate any false positives. From the screens, it was found that positively charged substitutions in the exposed hydrophobic patch region (sites 27, 29, 40, 50 & 52) are especially productive. In fact, one substitution, F50R, improves the purification yield to nearly the same level as wild-type sBBI. Productive amino acid substitutions were combined to select for the variant with the best overall yield after purification. Several variants were obtained with higher purification yields than even sBBI. The octuple variants, A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T and A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q, are particularly productive having greater than a five fold increase in final purification yield over the parent.
Collier
Katherine D
KD
Genencor International, A Division of Danisco, Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA.
Vogtentanz
Gudrun
G
Amin
Neelam S
NS
Estabrook
Melodie
M
Estell
David A
DA
Fox
Bryan
B
Power
Scott D
SD
Rao
Roopali
R
Schmidt
Brian F
BF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004220:Disulfides; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011995:Recombination, Genetic; D016415:Sequence Alignment; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D004220:Disulfides; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D042461:Vascular Endothelial Growth Factor A