Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Summary

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Abstract

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 10⁹-10¹⁰ obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel's method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

Introduction

mAbs have broad applications ranging from basic research to disease diagnostics and therapeutics. As of 2016, more than 60 mAbs have been approved by the United States Food and Drug Administration (USFDA) for clinical treatment of autoimmune diseases, cancer, and infectious diseases^1,2.

In 1975, Kohler and Milstein reported a technique for the continuous generation of antibodies of a single clonal specificity from a cellular source referred to as 'hybridomas' and this technique has subsequently become a cornerstone in medicine and industry^3,

Protocol

NOTE: Filter sterile tips must be used throughout when dealing with phage to avoid contamination to pipette gun and surrounding area. Aseptic area or hood must be used when handling with bacteria and phage experiments. Phage experiment area must be cleaned up using 2% sodium dodecyl sulfate (SDS) followed by 70% ethanol to avoid phage contamination. For making serial dilutions in this protocol, new tips should be used for each dilution.

1. E. Coli SS320 Electro-competent Cell Preparation

1. Pre-warm LB/tet agar plate (prepared and stored at 4 °C for less than 1-week old) at 37 °C incubator for 1 h. Use a sterile ....

Results

Following the flow chart of the Fab library construction (see Figure 1), we prepared M13KO7 helper phage pre-infected E. coli SS320 electro-competent cells. The efficiency of these electro-competent cells is estimated as 2 X 10⁹ cfu/µg when the Fab phagemid backbone for library construction was used (Figure 4).

The uracil incorporation efficiency by .......

Discussion

To construct high diversity, phage-displayed Fab libraries, quality control check points are needed to monitor various stages of the construction process, including the competency of electro-competent cells, quality of the dU-ssDNA template, efficiency of CCC-dsDNA synthesis, titer after electroporation, Fab folding, and amino acid diversity of CDRs by sequence analysis of Fab-phage clones.

High yield and purity of dU-ssDNA is essential for high mutagenesis rate. In our experience, phage induc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
1.0 M H3PO4	Fisher	AC29570
1.0 M Tris, pH 8.0	Invitrogen	15568-025
10 mM ATP	Invitrogen	18330-019
100 mM dithiothreitol	Fisher	BP172
100 mM dNTP mix	GE Healthcare	28-4065-60	solution containing 25 mM each of dATP, dCTP, dGTP and dTTP.
3,3’,5,5’-tetramethylbenzidine (TMB)	Kirkegaard & Perry Laboratories Inc	50-76-02
50X TAE	Invitrogen	24710030
Agarose	Fisher	BP160
Carbenicillin, carb	Sigma	C1389	100 mg/mL in water,  0.22 μm filter-sterilize, work concentration: 100 μg/mL.
Chloramphenicol, cmp	Sigma	C0378	100 mg/mL in ethanol, 0.22 μm filter-sterilize, work concentration: 10 μg/mL.
EDTA 0.5 M, pH 8.0	Invitrogen	AM9620G
Granulated agar	VWR	J637-500G
H2O2 peroxidase substrate	Kirkegaard & Perry Laboratories Inc	50-65-02
K2HPO4	Sigma	795488
Kanamycin, kan	Fisher	AC61129	50 mg/mL in water,  0.22 μm filter-sterilize, work concentration: 50 μg/mL.
KH2PO4	Sigma	P2222
Na2HPO4	Sigma	94046
NaCl	Alfa Aesar	U19C015
Nanodrop	Fisher	ND2000C
NaOH	Fisher	SS256	! CAUTION NaOH causes burns.
NON-Fat Powdered Milk	Sangon Biotech	A600669
PEG-8000	Fisher	BP233
Protein A-HRP conjugate	Invitrogen	101123
QIAprep Spin M13 Kit	Qiagen	22704
QIAquick Gel Extraction Kit	Qiagen	28706
QIAquick PCR Purification Kit	Qiagen	28104
Recombinant Protein L	Fisher	77679
T4 DNA polymerase	New England Biolabs	M0203S
T4 polynucleotide kinase	New England Biolabs	M0201S
T7 DNA polymerase	New England Biolabs	M0274S
Tetracycline, tet	Sigma	T7660	50 mg/mL in water, 0. 22 μm filter-sterilize, work concentration: 10 μg/mL.
Tryptone	Fisher	0123-07-5
Tween-20	Sigma	P2287
Ultrapure glycerol	Invitrogen	15514-011
Uridine	Sigma	U3750	25 mg/mL in ethanol, work concentration: 0.25 μg/mL.
Yeast extract	VWR	DF0127-08
Name	Company	Catalog Number	Comments
Strains
E.coli CJ236	New England Biolabs	E4141	Genotype: dut-  ung-  thi-1 relA1 spoT1 mcrA/pCJ105(F' camr). Used for preparation of dU-ssDNA.
E.coli SS320	Lucigen	60512	Genotype: [F'proAB+lacIq lacZΔM15 Tn10 (tetr)] hsdR mcrB araD139 Δ(araABC-leu)7679 ΔlacX74 galUgalK rpsL thi. Optimized for high-efficiency electroporation and filamentous bacteriophage production.
M13KO7	New England Biolabs	N0315S
Name	Company	Catalog Number	Comments
Equipment
0.2-cm gap electroporation cuvette	BTX
96-well 2mL Deep-well plates	Fisher	278743
96-well Maxisorp immunoplates	Nunc	151759
Baffled flasks	Corning
Benchtop centrifuge	Eppendorf	5811000096
Centrifuge bottles	Nalgene
ECM-630 electroporator	BTX
Magnetic stir bars	Nalgene
Thermo Fisher centrifuge	Fisher
High speed shaker	TAITEK	MBR-034P
Microplate shaker	QILINBEIER	QB-9002
Liquid handler for 96 and 384 wells	RAININ
Mutil-channel pipette	RAININ	E4XLS
Amicon concentrator	Merck	UFC803096