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Peptide Library Design Tools

An overlapping peptide library can be used for linear or continuous epitope mapping, which can be used to figure out which part of a given protein or peptide contains the essential amino acids that contribute to its functionality. Characterized by two parameters, peptide length and offset number, each library is generated by dividing the original protein or peptide into many overlapping peptides of equal length. Optimum peptide length is 8 to 20 amino acids, and as a general guideline, a peptide must be at least six residues in length for it to cover an epitope. The offset number is the number of amino acid residues shared by adjacent peptides, and it reflects the degree of overlap.

Careful selection of the offset number and the peptide length can minimize experiment cost, while maximizing data value. The offset number is usually designed to be 1/3 of the peptide length. Long peptides will generate more epitope hits per peptide, but they are difficult to synthesize and the library will contain fewer peptides. Shorter peptides are easier and cheaper to synthesize, but will result in fewer epitope hits per peptide. The combination of low offset number and short peptide length generates the largest number of peptides, while the combination of high offset number and long peptide length produces the least number of peptides.

Design Tool
Problem Causes Solutions
Wrong antibiotic was used or antibiotic concentration was too high
  • Ensure the correct antibiotic was applied to plates.
  • Use only concentration recommended by competent cell or antibiotic manufacturer.
Competent cell viability is low
  • Thaw competent cells on ice and use immediately.
  • Check expiration date of cells.
  • Do not re-freeze cells.
  • Do not vortex cells - gently tap to mix.
Few or no colony
transformants
 DNA insert encodes protein that is toxic to cells
  • Use a lower incubation temperature (25 – 30°C).
  • Use a cell strain and vector designed for tightly controlled transcription.
Heat-shock incubation too long
  • Reduce incubation time from 45 to 25 seconds.
Construct is too big
  • Use electroporation for vectors over 10 kb.
Too much ligation mixture was used for the transformation Ligation reaction components can inhibit transformation.
  • Dilute ligation reaction with TE buffer (up to 5 times).
Too much DNA in reaction
  • Use no more than 1-10 ng of DNA in 5 µl for a 100 µl reaction or in 1-3 µl for a 50 µl reaction.
Low ligation efficiency
  • Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10.
  • Use a DNA concentration of 1-10 µg/ml.
Construct recombined with genomic DNA
  • Switch to a Rec A- cell strain.
No Plasmid in
Colony tranformants
 Antibiotic concentration too low
  • Use antibiotic concentration recommended by manufacturer.
Antibiotic is degraded
  • Aliquot working volumes of antibiotic and avoid freeze-thaw cycles.
  • Add antibiotic to liquid plate media after sufficient cooling.
No insert in colony
tranformants plasmids
 Vector re-ligation
  • Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10. Use a DNA concentration of 1-10 µg/ml.
  • Dephosphorylate DNA with phosphatase to prevent re-ligation.
Sequencing of tranformants
plasmid reveals wrong
plasmid sequence
 DNA insert encodes protein that is toxic to cells
  • Use a lower incubation temperature (25 – 30°C).
  • Use a cell strain and vector designed for tightly controlled transcription.
Mutations introduced by initial PCR
  • Use a high-fidelity polymerase.
Inconclusive sequencing artifacts
  • Repeat sequencing reaction.
Transformation Protocol

Applications of Overlapping Library

Scan an antigen sequence for epitopes

Screen a protein for a substrate

Identify a T-cell epitope

Stimulate T-cells in T-cell assays

Map antibody epitopes

Search for other binding sites within a given protein

overlapping library
Overlapping Peptide
overlapping library
  
Alanine Scanning
alanine scanning library
     
Truncation Library
truncation library
  
Positional Scanning
positional scanning library
     
Random Library
random library
  
Scrambled Library
scrambled library
     
T Cell Truncated Library
T tell truncated tibrary
    

References

  • Sospedra M, Pinilla C, and Martin R. Use of combinatorial peptide libraries for T-cell epitope mapping. Methods. Mar 2003; 29(3): 236-47
  • Gershoni JM, Roitburd-Berman A, Siman-Tov DD, Tarnovitski Freund N, and Weiss Y. Epitope mapping: the first step in developing epitope-based vaccines. BioDrugs. 2007; 21(3): 145-56
  • Hemmer B, Pinilla C, Appel J, Pascal J, Houghten R, and Martin R. The use of soluble synthetic peptide combinatorial libraries to determine antigen recognition of T cells. J. Pept. Res. Nov 1998; 52(5): 338-45
  • Sung MH, Zhao Y, Martin R, and Simon R. T-cell epitope prediction with combinatorial peptide libraries. J. Comput. Biol. 2002; 9(3): 527-39
  • Paulmurugan R, and Gambhir SS. Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions. Anal. Chem. Mar 2007; 15; 79(6): 2346-53

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