RESUMO
The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Fator de Transcrição Associado à Microftalmia/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Endopeptidase K/metabolismo , Proteínas de Escherichia coli/genética , Zíper de Leucina , Proteínas de Membrana Transportadoras/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM ß-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in T(m), displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T(m) increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information.
Assuntos
Evolução Molecular Direcionada/métodos , Deleção de Sequência , beta-Lactamases/química , beta-Lactamases/genética , Estabilidade Enzimática , Biblioteca Gênica , Guanidina/farmacologia , Modelos Moleculares , Conformação Proteica , Desdobramento de Proteína/efeitos dos fármacos , Proteólise , Temperatura , Termodinâmica , beta-Lactamases/metabolismoRESUMO
We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.
Assuntos
Anticorpos Biespecíficos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Desenvolvimento de Medicamentos/métodos , Fragmentos Fab das Imunoglobulinas/farmacologia , Engenharia de Proteínas , Sequência de Aminoácidos/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/ultraestrutura , Becaplermina/antagonistas & inibidores , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Concentração Inibidora 50 , Injeções Intravítreas , Masculino , Modelos Moleculares , Estudo de Prova de Conceito , Conformação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM ß-lactamase (ßL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and ßL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the ßL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split ßL fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Engenharia de Proteínas/métodos , Mapeamento de Interação de Proteínas , beta-Lactamases/metabolismo , Arginina/genética , Arginina/metabolismo , Clonagem Molecular/métodos , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the geneIII protein into phage particles. We investigated functionality and display level of fluorescent proteins depending on the translocation pathway, which was the TAT, general secretory (SEC) or signal recognition particle (SRP) pathway mediated by the TorA, PelB or DsbA signal sequences, respectively. Importantly, for green fluorescent protein, yellow fluorescent protein and cyan fluorescent protein, only TAT, but not SEC or SRP, translocation led to fluorescence of purified phage particles, although all three proteins could be displayed regardless of the translocation pathway. In contrast, the monomeric red fluorescent protein mCherry was functionally displayed regardless of the translocation pathway. Hence, correct folding and fluorophor formation of mCherry is not limited to the cytosol. Furthermore, we successfully displayed firefly luciferase as well as an 83 kDa argonaute protein, both containing free cysteines. This demonstrates broad applicability of the TAT-mediated phagemid system for the display of proteins requiring cytoplasmic factors for correct folding and should prove useful for the display of proteins requiring incorporation of co-factors or oligomerization to gain function.
Assuntos
Bacteriófagos/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Dobramento de Proteína , Sobrevivência Celular , Clonagem Molecular , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Espaço Intracelular/metabolismo , Proteínas Luminescentes , Proteínas de Membrana Transportadoras/genética , Plasmídeos/genética , Via Secretória , Partícula de Reconhecimento de Sinal/genética , Partícula de Reconhecimento de Sinal/metabolismo , TemperaturaRESUMO
Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.