RESUMEN
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.
Asunto(s)
Clonación Molecular/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Inhibidor de la Tripsina de Soja de Bowman-Birk/aislamiento & purificación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Bacillus subtilis/genética , Sitios de Unión/genética , Disulfuros , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Recombinación Genética , Alineación de Secuencia , Inhibidor de la Tripsina de Soja de Bowman-Birk/biosíntesis , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/genéticaRESUMEN
A novel beta-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (beta-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual beta-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound beta-lactamase activity. SKBR3 cell-binding beta-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected beta-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted beta-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy.
Asunto(s)
Biblioteca de Péptidos , Profármacos/química , beta-Lactamasas/química , Secuencia de Aminoácidos , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Enterobacter cloacae/enzimología , Enterobacter cloacae/genética , Vectores Genéticos/genética , Humanos , Ligandos , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por Sustrato , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).