Skip to main content
OSTI.GOVU.S. Department of Energy Office of Scientific and Technical Information
U.S. Department of Energy
Office of Scientific and Technical Information
 

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

Journal Article · · PLoS ONE
 [1];  [2];  [3];  [4];  [5]
  1. Georg-August Universität Göttingen (Germany). Inst. of Microbiology and Genetics. Dept. of Molecular Microbiology and Genetics; Georg-August Universität Göttingen (Germany). Inst. of Microbiology and Genetics. Dept. Molecular Genetics and Preparative Molecular Biology; DOE/OSTI
  2. Georg-August Universität Göttingen (Germany). Inst. of Microbiology and Genetics. Dept. Molecular Genetics and Preparative Molecular Biology; Univ. Medical Center, Göttingen (Germany); Information Network of Depts. of Dermatology (IVDK), Göttingen (Germany)
  3. Los Alamos National Lab. (LANL), Los Alamos, NM (United States). Group T-g. Theoretical Biology and Biophysics; Univ. Greifswald (Germany). Inst. for Mathematics and Informatics; Georg-August-Universität Göttingen (Germany). Neurobiology Lab.
  4. Georg-August-Universität Göttingen (Germany). Neurobiology Lab.; German Primate Center GmbH, Göttingen (Germany). Neurobiology Lab.
  5. Georg-August Universität Göttingen (Germany). Inst. of Microbiology and Genetics. Dept. Molecular Genetics and Preparative Molecular Biology; Georg-August Universität Göttingen (Germany). Inst. of Microbiology and Genetics. Dept. Molecular Genetics
+ Show Author Affiliations

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 sublibraries, 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.

Research Organization:
Los Alamos National Laboratory (LANL), Los Alamos, NM (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC52-06NA25396
OSTI ID:
1627763
Journal Information:
PLoS ONE, Journal Name: PLoS ONE Journal Issue: 9 Vol. 10; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English

References (43)

Structural determinants in the sequences of immunoglobulin variable domain 1 1Edited by A. R. Fersht journal May 1998
A heterodimeric coiled-coil peptide pair selected in vivo from a designed library- versus -library ensemble 1 1Edited by A. R. Fersht journal January 2000
Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides journal February 2000
Protein surface amino acid compositions distinctively differ between thermophilic and mesophilic bacteria journal June 2001
Type II restriction endonucleases: structure and mechanism journal March 2005
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding journal May 1976
Analysis of Catalytic Residues in Enzyme Active Sites journal November 2002
Removing the Redundancy From Randomised Gene Libraries journal August 2003
Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins journal September 2003
Designing Repeat Proteins: Modular Leucine-rich Repeat Protein Libraries Based on the Mammalian Ribonuclease Inhibitor Family journal September 2003
Polishing the craft of genetic diversity creation in directed evolution journal December 2013
Advances in generating functional diversity for directed protein evolution journal February 2009
Directed Evolution of Streptavidin Variants Using In Vitro Compartmentalization journal September 2008
Anticalins: Exploiting a non-Ig scaffold with hypervariable loops for the engineering of binding proteins journal November 2013
High-throughput Generation of Synthetic Antibodies from Highly Functional Minimalist Phage-displayed Libraries journal November 2007
The Human Combinatorial Antibody Library HuCAL GOLD Combines Diversification of All Six CDRs According to the Natural Immune System with a Novel Display Method for Efficient Selection of High-Affinity Antibodies journal February 2008
Exploring the Structure–Function Loop Adaptability of a (β/α)8-Barrel Enzyme through Loop Swapping and Hinge Variability journal August 2011
Catalytic Versatility, Stability, and Evolution of the (βα) 8 -Barrel Enzyme Fold journal November 2005
Reducing Codon Redundancy and Screening Effort of Combinatorial Protein Libraries Created by Saturation Mutagenesis journal June 2012
Conformations of immunoglobulin hypervariable regions journal December 1989
Protein folding and misfolding journal December 2003
Programming cells by multiplex genome engineering and accelerated evolution journal July 2009
Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity journal February 2005
Construction of semi-randomized gene libraries with weighted oligonucleotide synthesis and PCR journal June 2006
A new and convenient approach for the preparation of β-cyanoethyl protected trinucleotide phosphoramidites journal January 2012
Mixed oligonucleotides for random mutagenesis: best way of making them journal January 2012
Efficient and flexible access to fully protected trinucleotides suitable for DNA synthesis by automated phosphoramidite chemistry journal January 1996
Rapid discovery of protein interactions by cell-free protein technologies journal October 2007
ProxiMAX randomization: a new technology for non-degenerate saturation mutagenesis of contiguous codons journal September 2013
Designed to be stable Crystal structure of a consensus ankyrin repeat protein journal February 2003
Imidazole Glycerol Phosphate Synthase from Thermotoga maritima: QUATERNARY STRUCTURE, STEADY-STATE KINETICS, AND REACTION MECHANISM OF THE BIENZYME COMPLEX journal March 2001
Human Combinatorial Fab Library Yielding Specific and Functional Antibodies against the Human Fibroblast Growth Factor Receptor 3 journal July 2003
Large-Scale Solid-Phase Preparation of 3′-Unprotected Trinucleotide Phosphotriesters - Precursors for Synthesis of Trinucleotide Phosphoramidites journal October 2000
Thermophilic Adaptation of Proteins journal January 2001
Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis journal January 1994
A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries journal October 1996
TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons journal September 2009
Combinatorial codon-based amino acid substitutions journal November 2004
To get what we aim for - progress in diversity generation methods journal June 2013
Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries journal November 2007
Structural Evidence for Evolution of the beta /alpha Barrel Scaffold by Gene Duplication and Fusion journal September 2000
Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library journal January 2007
Construction of “small-intelligent” focused mutagenesis libraries using well-designed combinatorial degenerate primers journal March 2012

Cited By (1)

Preparation of trinucleotide phosphoramidites as synthons for the synthesis of gene libraries journal January 2018

Figures / Tables (13)

Fig 1(p. 10)figure Fig 1
Fig 2(p. 11)figure Fig 2
Fig. 3(p. 12)figure Fig. 3
Table 1(p. 13)table Table 1
Table 2(p. 14)table Table 2
Fig 4(p. 15)figure Fig 4
Table 3(p. 16)table Table 3
Fig 5(p. 17)figure Fig 5
Fig. 6(p. 18)figure Fig. 6
Table 4(p. 19)table Table 4
Fig 7(p. 21)figure Fig 7
Table 5(p. 22)table Table 5
Fig. 8(p. 24)figure Fig. 8

Similar Records

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.
Journal Article · Mon Dec 31 23:00:00 EST 2001 · J. Mol. Biol. · OSTI ID:949678
POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS
Technical Report · Sun Dec 21 23:00:00 EST 2008 · OSTI ID:944546

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Science & Technology - Other Topics