Planning Your Peptide-Antibody Projects

PEPTIDE DESIGN PEPTIDE PURITY AND PEPTIDE SYNTHESIS OptimumAntigenTM DESIGN TOOL APPLICATION One of the axioms that GenScript respects is “Quality by Design”. Using GenScript can synthesize peptides with purity ranging from crude peptide up to 98%. Peptides created through GenScript's OptimumAntigenTM design program have many ELISA Titer OptimumAntigenTM design tool, GenScript's peptide designing process is in line with Here is a general guideline for peptide purity requirements: advantages over full length proteins when it comes to antibody production. Our O O TM this particular motto, delivering quality peptide and antibody projects smartly designed > 70% Immunological applications, polyclonal antibody production OptimumAntigen Design Tool combines the industry's most advanced algorithms with right from the beginning. Fmoc Protecting Group GenScript's time-tested expertise. Each peptide antigen is measured against several 74% ELISA tests NH C OH NH C > 85% Semi-quantitative enzyme-substrate studies protein databases to confirm the desired antibody and epitope specificity. GenScript 1. Peptide Purity Phosphorylation studies stands behind its peptide design and carrier protein conjugation technology. We 3.6% O > 95% In vitro bioassays such as ELISA, enzymology, biological activity ACTIVATION DEBLOCKING guarantee the delivery of effective antigens. For antibody generation and testing, peptide purity > 70% is enough, however, for Epitope mapping NH2 C OH biological activity studies, peptide purity > 95% is required. GenScript is able to develop > 98% cGMP peptides for drug studies Guaranteed Immune Response 22.4% peptides with different purity levels and has the ability to synthesize peptides with purity SAR studies 1 greater than 98%. O O GenScript stands behind the promise of its antigen design system. REPEAT TO 1:512.000 AMINO ACID STRUCTURES ≥1:32.000 2. Peptide Amino Acid Composition NH C NH2 C EXTEND THE If peptide antigens were designed, synthesized, and conjugated by GenScript，we O <1:20.000 Nonpolar, Aliphatic R Groups Polar, Uncharged R Groups CHAIN guarantee two positive clones for monoclonal antibodies or ELISA titer of 1:20,000 or Amino acid composition governs every aspect of the peptide’s functionality. Hydrophobic NH2 C OH better for polyclonal antibodies developed in any host. and hydrophilic characteristics are key factors to consider. The peptide should incorpo- (Gly or G) (Ala or A) (Ser or S) (Pro or P) H H O H H O H H O H H O COUPLING － － － － － － － － － －N+－－C －N+－－C －N+－－C － － － Based on our historical records, at least 96.4% of the orders (pie chart on the right) for rate antigenic amino acids sequence and avoid problematic amino acids such as H C H C H C H－N+－－C C Amino Acid 2 － － － － － － Strategies - - -

－

HHO HCHO H CH O － - － 3 OH CH CH O which peptide antigens designed, synthesized and conjugated by GenScript received , , and . － H 2 2 CH 2 ELISA titer (≥ 1:32,000) higher than guaranteed. (Val or V) (Leu or L) Our OptimumAntigenTM program searches and assesses your sequence to identify the H H O H H O (Thr or T) Cysteine (Cys or C) － － 3. Peptide Length － － － － + H H O O H－N －－C C H－N+－－C C O H H O best candidate peptides that optimize solubility and antigenicity and maximize the chance

－ － － － － － － + － － － HCHO- O- H－N －－C C + H CH2 H－N －－C C TM － － of synthesis success. Each peptide is compared with our curated BLAST databases of

－ -

Peptide length governs the level of difficulty when synthesizing peptides. For long and H C CH H CH O － － - OptimumAntigen Benefits 3 3 CH OH H CH O C 2 NH NH C Side-Chain － CH H C CH 3 － over two dozen species to limit cross-reactivity while improving overall antibody complex peptides, GenScript implements its proprietary recombinant peptide system and 3 3 SH Protecting Group 1 specificity. The following strategies are used for OptimumAntigenTM program: has the ability to produce peptides with lengths up to 200 residues. Peptide antigens are lsoleucine (Ile or I) Methionine (Met or M) (Asn or N) (Gln or G) • Strong antigenicity H H O H H O H H O H H O

－ － － － － recommended to contain 10-15 residues to be used in antibody production. － － － － － － FINAL DEBLOCK －N+－－C － + + • Avoid unavailable epitopes due to structural constraints H C H－N+－－C C H－N －－C C H－N －－C C • Sequence length － － － － － - － － － - - H CH O - O O O H CH2 H CH2 H CH2 CH CH － • Specify desirable cross-reactivity 2 3 －NH • Hydrophilic, surface-oriented, and flexible － C 2 CH2

4. Peptide Solubility SCH2 － H C － － 3 O － － CH O C NH2 • Eliminate re-synthesis times with built-in peptide tutorial for synthesis and solubility 3 • Targeting the N terminus or C terminus GenScript designs peptides of varied solubility by changing the frequency of occurrence Positively Charged R Groups Side-Chain • Guaranteed immune response • Continuous versus discontinuous epitopes Aromatic R Groups O O of hydrophobic and hydrophilic amino acids. Hydrophobic amino acid content is Protecting Group 2 (Lys or K) (His or H) (Arg or R) • Algorithms suggested to be kept below 50% with at least one charged residue incorporated within (Phe or F) (Tyr or Y) H H O H H O H H O NH2 C NH C － － － － － － － － － • Coupling strategy H H O H H O H－N+－－C C H－N+－－C C H－N+－－C C GenScriptGenScript aanimalnimal ffacilityacility iiss every five amino acids. In addition, a single or addition of polar － － － － － － － － + － － － － H－N+－－C C H－N －－C C HCHO- O- O- 2 H CH2 H CH2

－ － AAALACAAALAC InternationalInternational • Experience － － － residues to the N- or C-terminus may also improve peptide solubility. - O- － － H CH O H CH2 + 2 CH2 C N H CH2 － －

－ CH － INTERNATIONAL accredited.accredited. CH － + • Mechanism learning algorithms 2 HC N － NH CH CLEAVAGE/DEPROTECTION 2 2 － －

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－ H H N+－CH H N－C －NH 5. Secondary Structure OH 3 2 2 Tryptophan (Trp or W) Resin H H O Negatively Charged R Groups During peptide synthesis, beta sheet formation can cause incomplete solvation of the － － － O O H－N+－－C C － － O- (Asp or D) (Glu or E) growing peptide thus sequences in the final product. GenScript sequences avoid H CH2 － C －CH H H O H H O NH2 C NH C OH －

multiple or adjacent residues of Val, Ile, Tyr, Phe, Trp, Leu, Gln, or Thr to prevent beta － － － － NH － － H－N+－－C C H－N+－－C C sheet formation. If the above recommendation cannot be implemented practically, － － O- － － O- HCH2 H CH2

－ － FREE PEPTIDE C CH conservative replacement may help by reinserting a Gly or Pro at every third residue or － 2

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O O － Activator C－ replacing Gln with Asp, or Thr with Ser. -O O ANTIBODY PRODUCTION

Polyclonal Antibody Production Monoclonal Antibody Production NOMENCLATURE AND PROPERTY OF AMINO ACIDS PEPTIDE SOLUBILITY GUIDELINES Antigen Design GLNTRKKLQ GLNTRKKLQ Antigen Design

pKa Values The solubility of peptides varies depending on its amino acid sequence and modifications. Hydropathy Occurrence in Amino Acid 3-Letter Symbol 1-Letter Symbol M pK pK pK p Solubility test guideline: t 1 2 R I Index* Protein (%) H N+ H N+ 3 O R Peptide Synthesis 3 O R Peptide Synthesis －

－ GenScript Custom Peptides － －

－ － CH－－C NH－C－Cys － and Coupling CH－－C NH－C－Cys －KLH KLH and Coupling

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Nonpolar, Aliphatic R Groups － O O

Glycine Gly G 75 2.34 9.60 5.97 -0.4 7.2 Alanine Ala A 89 2.34 9.69 6.01 1.8 7.8 Calculate the charge of the peptide Valine Val V 117 2.32 9.625.96 . 4.2 6.6 Animal Leucine Leu L 131 2.36 9.60 5.98 3.8 9.1 Immunization Ile I 131 2.36 9.60 6.02 4.5 5.3 Animal Methionine Met M 149 2.28 9.21 5.74 1.9 2.3 < 0 (Acidic peptide) > 0 (Basic peptide) 0 (Neutral peptide) Immunization Aromatic R Groups Cell Fusion Add water Add water Phenylalanine Phe F 165 1.83 9.13 5.48 2.8 3.9 Add organic solvents Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 -1.3 3.2 (acetonitrile, methanol, isopropanol, etc.) Tryptophan Trp W 204 2.83 9.39 5.89 -0.9 1.4 a. Cys-containing peptides Antiserum + + + + +

Add NH4OH (< 50 μl) b. Very hydrophobic peptides Add 10%-30% acetic acid +

Preparation + Hybridoma

+

Polar, Uncharged R Groups

+ c. Tend-to-aggregate peptides +

+ +

Screening

Serine Ser S 105 2.21 9.15 5.68 -0.8 6.8 abc Add TFA (< 50 μl) Proline Pro P 115 1.99 10.60 6.30 1.6 5.2 Add small Add small Add 6 M guanidine•HCl Threonine Thr T 119 2.09 9.10 5.60 -0.7 5.9 amount DMF amount DMSO or 8 M urea Cysteine Cys C 121 1.96 10.28 8.18 5.07 -2.5 1.9 Asparagine Asn N 132 2.02 8.80 5.41 -3.5 4.3 Purification Antibody Glutamine Gln Q 146 2.17 9.13 5.65 -3.5 4.2 Production Dilute to the desired concentration Positively Charged R Groups Roller Bottle Cell Culture / Ascites Production

Western Blot / ELISA / Lysine Lys K 146 2.18 8.95 10.53 9.74 -3.9 5.9 Peptide Solution Histidine His H 155 1.82 9.17 6.00 7.59 -3.2 2.3 IHC / IP / Others Arg Arginine R 174 2.17 9.04 12.48 10.76 -4.5 5.1 Antibody Product

Negatively Charged R Groups Peptide Stock Solution1

Aspartic acid Asp D 133 1.88 9.60 3.65 2.77 -3.5 5.3 Peptide Storage2 Glutamic acid Glu E 147 2.19 9.67 4.25 3.22 -3.5 6.3 Note: GenScript USA Inc. Tel: 1-732-885-9188

1. It is recommended that the concentration of the stock solution be approximately 1-2 mg of peptide per ml of solution. This is dilute enough to minimize precipitation during storage but concentrated enough to 120 Centennial Ave. Email: [email protected] * A scale combining hydrophobicity and hydrophilicity of R groups, it can be used to measure the tendency of amino acid to seek an aqueous environment (- values) allow relatively small volumes (< 100 μl) of aliquot to be used in assays, thereby minimizing the effects of the solvent. Piscataway, NJ 08854, USA Web: www.genscript.com or a hydrophobic environment (+ values). 2. Lyophilized peptides will remain stable for over one year if stored at -20°C. We recommend aliquoting dissolved peptide into tubes for storage at or below -20°C. It is recommended that peptides containing methionine, cysteine, or tryptophan residues be stored in an oxygen-free environment. Your Innovation Partner in Drug Discovery!

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