Protein Variant Libraries

Sequence libraries are a powerful tool for structure-function relationship analysis, or modifying the activities of proteins or DNA regulatory regions. DNA2.0 makes libraries in several formats to meet different research needs. Libraries are delivered ready for screening to identify variants with altered function.

Protein Engineering for Improved Functionality
Researchers use large libraries, focused libraries and rational design to engineer protein function. Success is largely a matter of trying over and over again. Typically, the process illustrated below is repeated until significant improvements are obtained.

Protein Variant Libraries - Protein Engineering Process

Read more:
Nature Methods 2011. 8: 623. Protein engineering: navigating between chance and reason. Monya Baker.
Genetic Engineering & Biotechnology News 2013. 33(8):18. Library Format for Bioengineering. Maximizing Screening Efficiency through Good Design. Levay-Young et al.

Designing a Library
It is important when designing protein variant libraries to carefully evaluate the number of unique sequences thoretically contained in the library. For most applications the transformation frequency provides a practical upper limit on the number of sequences that can be assessed. The methods used to measure protein function may further reduce this number.

How to calculate and assess library size.

Use the Library Designer to start your library today.

I. Antibody Libraries:
The antigen-binding site of an antibody is composed of six complementarity determining regions (CDRs) or hypervariable regions—three within the light-chain variable domain (VL) and three within the heavy chain variable domain (VH). Harness the power of synthetic biology to create antibody libraries with diversity in one or more CDRs. Libraries with diversity up to 1010 can be created. Precisely control diversity and choose frameworks with favorable properties.

Synthetic antibody libraries are constructed using oligonucleotides to introduce complete or tailored degeneracy into CDRs of one or more V genes. Synthetic diversity bypasses natural biases and redundancies of antibody repertoires created in vivo and allows control over the genetic makeup of V genes and the introduction of diversity.

IA. Deep Mutational Scanning of Erbitux Antibody CDR

DNA2.0 synthesized ∼2,000 unique Erbitux mAb genes for researchers at AbbVie Biotherapeutics Corp, encompassing 59 individual CDR positions, each with every possible single amino acid degeneracy. The resulting sequence-function correlation method can be used to engineer antibody functionality (specificity, immunogenicity, stability, aggregation, etc.) with high resolution and predictability.
mAbs 2013, 5(4):523-32. Forsyth et al. AbbVie Biotherapeutics Corporation.

FACS sorting of 293c18 cells expressing surface-displayed hu225 and libraries

FACS sorting of 293c18 cells expressing surface-displayed hu225 and libraries. (A) Wild type hu225 and (B-C) hu225 VH and VL libraries, respectively, were stained with AF647-EGFR-Cλ fusion protein and anti-human kappa PE (to normalize for IgG surface expression). Approximately 108 cells from each library were sorted on a MoFlo MLS instrument using high affinity binding (Bind) and expression (Exp) gates are indicated.

IB. Adimab Antibody Libraries

DNA2.0 has made several antibody libraries for Adimab, Inc, a pioneer in the integration of antibody discovery, maturation and bio-manufacturing. Adimab uses libraries that recreate the diversity contained within the natural human pre-immune repertoire by accounting for the recombination of the human immunoglobulin gene segments (i.e., V, D, and J).The result is a unique antibody library that can be readily screened to identify large numbers of IgGs specific to a particular target, with very broad sequence diversity and epitopic coverage. Visit Adimab to learn more.

Adimab Logo

Learn more about DNA2.0 Antibody Libraries.

II. Synthetic Circular Permutation Library:

Together with Stefan Lutz and Ashley Daugherty of Emory University, DNA2.0 applied novel library construction methods to engineer improved activity in Old Yellow Enzyme. Using a Circular Permutation (CP) library design, they were able to significantly enhance the function of the flavin-dependent oxidoreductase Old Yellow Enzyme (OYE1) from Saccharomyces pastorianus. OYEs are found in a variety of Biotech and Pharmaceutical applications, and are thus attractive candidates for increased catalytic properties. DNA2.0 synthesized a cpOYE library exhaustively creating 240 circular permutants that were screened rapidly and reliably against three substrates. Experimental analysis of the permutants identified 70 functional OYE1 variants with improved catalytic activity. The data revealed the importance of four distinct regions near the active site for improved enzyme performance. The use of a synthetic CP library “dramatically accelerated the process of identifying catalytically improved cpOYE variants for any substrate of choice.”

Circular Permutation Library Daugherty JACS

Primary screening data of cpOYE library for ene-reductase activity on three substrates (A, B and C). Whole-gene synthesis created a native cpOYE library with perfect distribution (red lines in inner circle). Catalytic activity of library members for each substrate was measured by semiquantitative assay and is reflected in the length of the green lines. Wild-type activity is indicated by the gray-shaded area. For all three substrates, four sectors (I−IV) in the protein sequence with activity equal to or better than that of wild type were identified.

Learn More: JACS 2013, 135(38):14425-32. Daugherty et al.

II. Improving Protein Yield using a Combinatorial Library:
The Bowman-Birk protease inhibitors (BBI) are plant protein scaffolds to present binding peptides selected by established methods (e.g. phage display). Scientists at Genencor, generated site-saturation libraries to improve protein yield by 5 fold.

Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduced the overall purification yield. To improve production yields, scientists at Genencor, generated site-saturation libraries at 39 out of 66 amino acid residues of BBI-AV. Libraries were screened to select desired variants. Productive amino acid substitutions were found and combined to select for the variant with the best overall yield after purification. An octuple variant with greater than five- fold increase in final purification yield over the parent, was obtained. Several variants were obtained with higher purification yields than even sBBI. Protein Expr Purif 2009 Collier, et al.

Learn more about DNA2.0 Combinatorial Libraries.

Webinar: Design of Protein Variant Libraries to Examine Sequence Diversity

Protein variant libraries can allow exploration of "sequence space" over modest to very large areas. We discuss the basic library types, their strengths and possible uses. Use of single site saturation and combinatorial libraries to improve enzyme properties and Antibody libraries to increase antibody affinity are specific examples. Basic information required for DNA2.0 to provide useful Quotes for library synthesis is also discussed. April 5, 2012
View a pdf of the webinar slides.

DNA2.0 Presentations and White Papers:

Consult with a Library Design Specialist Contact DNA2.0 at +1 877 362 8646 or info@DNA20.com to determine the best possible option for your research project.

Custom Design Your Own Alanine Scanning Library Use the DNA2.0 Library Designer to start your library today.

Variant Synthesis: If your variant project is less complex than a library, just a few synthetic gene variants, see Variant Synthesis for how DNA2.0 can create the ideal strategy to move your research forward quickly and affordably.

When screening a large library becomes impractical, a rational, design of experiment (DoE) methodology can be used to create variant sets that thoroughly sample a defined set of substitutions in a minimal number of test genes. Learn more.

Research Question Solution

How can I test BioBrick modules or genetic elements?

Modular Library

Protein Variant Libraries - Modular

How can I increase the affinity or specificity of my antibody?

Antibody Library

Protein Variant Libraries - Antibody Library mini image

What effect do changes at a single site have on function?

Single Site Saturation Library

Protein Variant Libraries - Site Scanning

How can individual changes be combined to improve function?

Combinatorial Site Library

Protein Variant Libraries - Combinatorial library mini image

Additional Libraries

Alanine Scanning
Random Mutagenesis
Truncation

Consult with a Library Design Specialist Today

To determine the best possible option for your research project please contact our Library Design Specialists at +1 877 362 8646 or info@DNA20.com.
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