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A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

ABSTRACT: A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

SUBMITTER: Popova B

PROVIDER: S-EPMC4565649 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

Popova Blagovesta B Schubert Steffen S Bulla Ingo I Buchwald Daniela D Kramer Wilfried W

PloS one 20150910 9

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermoto ...[more]

PMID: 26355961

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Project description:We describe a new method for encoded synthesis, efficient on-resin screening, and rapid unambiguous sequencing of combinatorial peptide libraries. An improved binary tag system for encoding peptide libraries during synthesis was designed to facilitate unequivocal assignment of isobaric residues by MALDI-TOF MS analysis. The improved method for encoded library synthesis was combined with a new versatile on-resin screening strategy that permitted multiple stages and types of screening to be employed successively on one library under mild conditions. The new method facilitated a combinatorial study of transglutaminase (TGase) enzyme substrate peptides, revealing new details of the effect of amino acid composition on TGase substrates. The approach was first demonstrated for an encoded library (130,321 compounds) of lysine pentapeptide substrates of TGase, synthesized using the "split-mix" method. The library was reacted on-resin with TGase enzyme and a soluble desthiobiotin labeled glutamine substrate. Initial screening was performed by adsorbing streptavidin-coated magnetic microparticles onto library beads, followed by magnetic separation. The differential binding affinities of desthiobiotin and biotin for streptavidin were exploited to release the magnetic microparticles and regenerate the desthiobiotin-labeled resin beads for further screening by flow-cytometry-based automated bead sorting, resulting in 345 beads that were sequenced by MALDI-TOF MS analysis. A second library consisted of encoded glutamine hexapeptide substrates, which was reacted on-resin with TGase enzyme and a soluble desthiobiotin-labeled cadaverine. Two-stage screening identified 267 glutamine peptides as TGase-reactive, of which 21 were further analyzed by solution-phase enzyme kinetics. Kinetic results indicated that the peptide PQQQYV from the library has a 68-fold greater substrate specificity than the best known glutamine substrate QQIV. The new encoding and screening strategies described here are expected to be broadly applicable to synthesis and screening of combinatorial peptide libraries in the future.

Dataset Information

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

Publications

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

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