噬菌体展示肽库说明书

噬菌体展示肽库说明书
噬菌体展示肽库说明书

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Ph.D.? Phage Display Libraries

Instruction Manual neB #e8100S, #e8101S, #e8102L,

#e8110S, #e8111L, #e8120S, #e8121L

Store at –20°C

table of Contents:

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Media and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 General M13 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Construction of pIII-Display Libraries using M13KE . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 Panning Protocols

Protocol 1: Surface Panning Procedure (Direct Target Coating) . . . . . . . . .14

Protocol 2: Solution-phase Panning with a Biotinylated Target

and Streptavidin Plate Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

Protocol 3: Solution-phase Panning with Affinity Bead Capture . . . . . . . . .19 Post Panning Protocols

Plaque Amplification for ELISA or Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Sequencing of Phage DNA

Rapid Purification of Sequencing Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Phage ELISA Binding Assay with Direct Target Coating . . . . . . . . . . . . . . . . . . .25

Use of Synthetic Peptides in Specificity Analysis . . . . . . . . . . . . . . . . . . . . . . . . . .27 Expression of Selected Sequences as Monovalent MBP Fusions . . . . . . . . . . . . . . . .27 Appendix:

Optimizing Peptide Binding Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Choice of Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30

Selection of Cell-Specific Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

1

Kit Components

All kit components should be stored at –20°C except where noted:

Phage Display Peptide Library

100 μl, ~ 1 x 1013 pfu/ml . Supplied in TBS with 50% glycerol .

-96 gIII sequencing primer

5′- HO CCC TCA TAG TTA GCG TAA CG –3′, 100 pmol, 1 pmol/μl

-28 gIII sequencing primer

5′- HO GTA TGG GAT TTT GCT AAA CAA C –3′, 100 pmol, 1 pmol/μl

E. coli ER2738 host strain

F′ proA+B+ lacI qΔ(lacZ)M15 zzf::Tn10 (Tet R)/fhuA2 glnV thi Δ(lac-proAB)

Δ(hsdMS-mcrB)5 (rk– mk– McrBC–) . Host strain supplied as 50% glycerol cul-ture; not competent . Store at –70°C .

Streptavidin, lyophilized

1 .5 mg

Biotin, 10 mM

100 μl

Ph .D . Peptide Display Cloning System (Supplied with E8101 only)

20 μg M13KE glll Cloning Vector, 2150 pmol Extension Primer Introduction

Phage display describes a selection technique in which a library of peptide

or protein variants is expressed on the outside of a phage virion, while the

genetic material encoding each variant resides on the inside (1-3) . This creates

a physical linkage between each variant protein sequence and the DNA encod-

ing it, which allows rapid partitioning based on binding affinity to a given target molecule (antibodies, enzymes, cell-surface receptors, etc .) by an in vitro

selection process called panning (4) . In its simplest form, panning is carried out by incubating a library of phage-displayed peptides on a plate (or bead) coated with the target, washing away the unbound phage, and eluting the specifically bound phage (Figure 1) . The eluted phage are then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding se-quences . After 3–4 rounds, individual clones are characterized by DNA sequenc-ing and ELISA .

The Ph .D .? (phage display) system is based on a simple M13 phage vector,

modified for pentavalent display of peptides as N-terminal fusions to the minor coat protein pIII (5-7) . This protein modulates phage infectivity by binding to the F-pilus of the recipient bacterial cell, and is present in 5 copies clustered at one end of the mature M13 virion (2,8) . If the displayed peptide is sufficiently short (< 50 residues), the infectivity function of pIII is not affected, and all 5 copies can carry displayed peptides without measurable attenuation of phage infectiv-ity (6) . As a result, the M13KE genome contains only a single copy of gene III

2

Panning with a pentavalent peptide library displayed on pIII.

3

4The premade libraries (Figure 2) consist of linear heptapeptide (Ph .D .-7) and dodecapeptide (Ph .D .-12) libraries, as well as a loop-constrained heptapeptide (Ph .D .-C7C) library . The randomized segment of the Ph .D .-C7C library is flanked by a pair of cysteine residues, which are oxidized during phage assembly to a disulfide linkage, resulting in the displayed peptides being presented to the target as loops . All of the libraries have complexities on the order of 109 independent clones, which is sufficient to encode most if not all of the possible 7-mer (1 .28 x 109) peptide sequences, but only a tiny fraction (less than 1 millionth) of the 4 .1 x 1015 possible 12-mer sequences . The Ph .D .-12 library can thus be thought of as having the equivalent diversity of the Ph .D .-7 library, but spread out over 12 residues . In both the Ph .D .-7 and the Ph .D .-12 libraries, the first residue of the peptide-pIII fusion is the first randomized position, while the first randomized position in the Ph .D .-C7C library is preceded by Ala-Cys (Figure 2) . All of the libraries contain a short linker sequence between the displayed peptide and pIII: Gly-Gly-Gly-Ser .

See Appendix, page 30 for further discussion on choosing which library to use in a given experiment.Figure 2:

X 7GGGS X 12GGGS A C X 7C GGGS

SS

Ph.D.-7

Ph.D.-12

Ph.D.-C7C Experiments at New England Biolabs have identified consensus peptide bind-ing sequences against a variety of proteins including enzymes, cell-surface re-ceptors, and monoclonal antibodies (Figure 3) . In all cases the prepared librar-ies have been demonstrated to be of sufficient complexity to produce multiple DNA sequences encoding the same consensus peptide motifs . The system has been used elsewhere for myriad applications, including epitope mapping (11-15), anti-microbial/viral peptides (16-22), material-specific peptides (23-26), small molecule binders (9, 27-31) and novel enzyme substrates (32) . The Ph .D . libraries have been used extensively for discovery of bioactive peptides through in vitro and in vivo panning approaches: peptide antagonists of VEGF-mediated angiogenesis (33), plasmodesmal trafficking petides (34) and cell targeting peptides (35-40) have all been pulled from the Ph .D . librar-ies (35-41) . In a particularly dramatic application, the Ph .D .-12 library was panned against Taxol , and the selected sequences were compared to a protein database to identify the natural target for the drug as Bcl-2 (9) . This quite unexpectedly demonstrated that short peptides from an unstructured peptide library can mimic a three-dimensional ligand binding site, greatly increasing Peptide libraries available from NEB. Randomized segments and linker sequences are indicated.

Y G G F M T S E K Q T P …

Y G W I S P P L H L P T

Y Q P D N P S R Q I A N

Y W P A H I R A V P M I

R L D D I K N T L A F S

S S D V Y S L Y P F I M

E F F P H P M L H N S R D N W P Y R P S F S L S S H N T Y S A P R P S A S L L H Y A S S L S L M

F N Q N A E P F S S R P

H P R Q L L H H P L S P Y G G F L I G L Q D A S

Y G G F H Y K E T G A L

D D D 1st round sequences:

2nd round sequences:

3rd round sequences:β-endorphin:

Y G G F M T T P S H V P Y G G F M T T P S H V P Y G G F I S Q T Q H Y S Y G G F I S Q T Q H Y S Y G G F G N S L V M P V Y G G F S M P F L P A L Y G A F D V T T G V T S Y G V F N P H Y L P S L A P S T D K Q A T M P L A S V A V S S R Q D A A

6

Media and Solutions

LB Medium:

Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl . Autoclave, store at room temperature .

IPtG/Xgal Stock:

Mix 1 .25 g IPTG (isopropyl-β-D-thiogalactoside) and 1 g Xgal (5-Bromo-4-chloro-3-indolyl-β-D-galactoside) in 25 ml DMF (dimethyl formamide) . Solu-tion can be stored at –20°C .

LB/IPtG/Xgal Plates:

1 liter LB medium + 15 g/l agar . Autoclave, cool to < 70°C, add 1 ml IPTG/Xgal Stock per liter and pour . Store plates at 4°C in the dark .

SoC Medium (Ph.D. Cloning System only):

Per liter: 20 g Bacto-Tryptone, 5 g yeast extract, 0 .5 g NaCl . Dispense into 100 ml aliquots and autoclave . Prior to use, add 0 .5 ml 2 M MgCl 2 and 2 ml 1 M glucose (both sterile) per 100 ml . Store at 4°C .

top Agar:

Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 7 g Bacto-Agar (or eletrophoresis grade agarose) . Autoclave, dispense into 50 ml aliquots . Store solid at room temperature, melt in microwave as needed .

tetracycline Stock (suspension):

20 mg/ml in 1:1 Ethanol:Water . Store at –20°C . Vortex before using .

LB+tet Plates:

LB medium + 15 g/l Agar . Autoclave, cool to < 70°C, add 1 ml Tetracycline Stock and pour . Store plates at 4°C in the dark . Do not use plates if brown or black .

Blocking Buffer:

0 .1 M NaHCO 3 (pH 8 .6), 5 mg/ml BSA, 0 .02% NaN 3 (optional) . Filter sterilize, store at 4°C .

tBS:

50 mM Tris-HCl (pH 7 .5), 150 mM NaCl . Autoclave, store at room tempera-ture .

PeG/naCl:

20% (w/v) polyethylene glycol–8000, 2 .5 M NaCl . Autoclave, mix well to com-bine separated layers while still warm . Store at room temperature .

7

Iodide Buffer:

10 mM Tris-HCl (pH 8 .0), 1 mM EDTA, 4 M sodium iodide (NaI) . Store at room temperature in the dark . Discard if color is evident .

Streptavidin Stock Solution:

Dissolve 1 .5 mg lyophilized Streptavidin (supplied) in 1 ml 10 mM sodium phosphate (pH 7 .2), 100 mM NaCI, 0 .02% NaN 3 (optional) . Store at 4°C or –20°C (avoid repeated freezing/thawing) .

General M13 Methods

It is important to note that unlike phage lambda, M13 is not a lytic phage. Plaques are caused by diminished cell growth rather than cell lysis, and are turbid rather than clear. Plating on Xgal/IPTG media is strongly recommended to facilitate visualization of plaques.

Strain Maintenance

1 . The recommended E. coli host strain ER2738 (F′ proA +B + lacI q Δ(lacZ)M15 zzf::Tn10 (Tet R )/fhuA

2 glnV Δ(lac-proAB) Δ(hsdMS-mcrB)5 [r k – m k – McrBC –]) is a robust F + strain with a rapid growth rate and is particularly well-suited for M1

3 propagation . ER2738 is a recA + strain, but we have never observed spontaneous in vivo recombination events with M13 or phagemid vectors . Commercially available F + strains such as DH5αF′and XL1-Blue can probably be substituted for ER2738, but have not been tested with our vector system . Any strain used should be supE (GlnV ) in order to suppress amber (UAG) stop codons within the library with glutamine .

2 . Because M1

3 is a male-specific coliphage, it is recommended that all cultures for M13 propagation be inoculated from colonies grown on media selective for presence of the F-factor, rather than directly from the glycerol culture . The F-factor of ER2738 contains a mini-transposon which confers tetracycline resistance, so cells harboring the F-factor can be selected by plating and propagating in tetracycline-containing medium . Tetracycline does not need to be added to media during phage amplification .

3 . Streak out ER2738 from the supplied glycerol culture onto an LB+Tet plate . Invert and incubate at 37°C overnight and store wrapped with para-film at 4°C in the dark for a maximum of 1 month . For archival purposes, we recommend that fresh glycerol stocks of ER2738 prepared from liquid cultures (48) .

4 . ER2738 cultures for infection can be grown either in LB or LB+Tet media . Loss of F-factor in nonselective media is insignificant as long as cultures are not serially diluted .

Avoiding Phage Contamination

The library cloning vector M13KE differs from wild-type filamentous phage vector in that the lacZα-peptide cloning sequence (which permits blue/white screening) has been inserted in the vicinity of the (+) strand origin of replication, resulting in a longer replication cycle. In addition, display of foreign peptides as N-terminal fusions to pIII (which mediates infectivity by binding to the F-pilus of the recipient bacterium) may attenuate infectivity of the library phage relative to wild-type M13. As a result, there is the possibility of in vivo selection for any contaminating wild-type phage during the amplification steps between rounds

of panning. In the absence of a correspondingly strong in vitro binding selection, even vanishingly small levels of contamination can result in a majority of the phage pool being wild-type phage after three rounds of panning.

1 . The potential for contamination with environmental bacteriophage can be

minimized by using aerosol-resistant pipette tips and wearing gloves for all

protocols .

2 . Because the library cloning vector M13KE is derived from the common

cloning vector M13mp19, which carries the lacZα gene, phage plaques

appear blue when plated on media containing Xgal and IPTG . Environmental

filamentous phage will typically yield colorless plaques when plated on the

same media . These plaques are also larger and “fuzzier” than the library

phage plaques . We strongly recommend plating on LB/IPTG/Xgal plates

for all titering steps and, if white plaques are evident, picking ONLY blue

plaques for sequencing.

3 . Severe contamination (white plaques present in large numbers) can lead

to contamination of subsequent panning experiments . To prevent this, all

solutions should be re-autoclaved if possible; any solutions containing heat-

labile components should be re-made . The work area including incubators

should be wiped down with ethanol . Pipettors should be disassembled and

the parts soaked in detergent, rinsed carefully with sterile water, and reas-

sembled .

Phage titering

The number of plaques will increase linearly with added phage only when the multiplicity of infection (MOI) is much less than 1 (i.e., cells are in considerable excess). For this reason, it is recommended that phage stocks be titered by diluting prior to infection, rather than by diluting cells infected at a high MOI. Plating at low MOI will also ensure that each plaque contains only one DNA sequence.

1 . Inoculate 5–10 ml of LB with ER2738 from a plate and incubate with shak-

~ 0 .5) .

ing 4–8 hrs (mid-log phase, OD

600

2 . While cells are growing, melt Top Agar in microwave and dispense

3 ml

into sterile culture tubes, one per expected phage dilution . Maintain tubes at

45°C .

3 . Pre-warm, for at least one hour, one LB/IPTG/Xgal plate per expected dilu-

tion at 37°C until ready for use .

8

9

4 . Prepare 10 to 103-fold serial dilutions of phage in LB; 1 ml final volumes are convenient . Suggested dilution ranges: for amplified phage culture super-natants, 108–1011; for unamplified panning eluates, 101–104 . Use aerosol-resistant pipette tips to prevent cross-contamination, and use a fresh pipette tip for each dilution .

5 . When the culture in Step 1 reaches mid-log phase, dispense 200 μl into microfuge tubes, one for each phage dilution .

6 . To carry out infection, add 10 μl of each phage dilution to each tube, vortex quickly, and incubate at room temperature for 1–5 minutes .

7 . Transfer the infected cells one infection at a time to culture tubes containing 45°C Top Agar . Vortex briefly and IMMEDIATELY pour culture onto a pre-warmed LB/IPTG/Xgal plate . Gently tilt and rotate plate to spread top agar evenly .

8 . Allow the plates to cool for 5 minutes, invert, and incubate overnight at 37°C .

9 .

Count plaques on plates that have approximately 100 plaques . Multiply each number by the dilution factor for that plate to get phage titer in plaque form-ing units (pfu) per 10 μl .Storage of Phage Solutions

Panning experiments may be interrupted at several points in the protocol. Phage in suspen-sion with NaCl/PEG may be stored for several weeks at 4°C. Eluted phage in neutralized buf-fer may be stored at 4°C for up to 1 week. Amplified phage may be stored in neutral buffer for up to 3 weeks with, either the addition of 0.02% NaN 3 or incubation at 65°C for 15 min to kill residual E. coli. Amplified phage may be stored long term, 5 years or more, by adding an equal volume of sterile glycerol, vortexing and placing at –20°C. It is not necessary to store phage at temperatures below –20°C, however, if required, single use aliquots of phage stocks may be flash frozen and thawed once without significant loss of titer.

Construction of pIII-Displayed Peptide Libraries using M13Ke (Ph.D. Cloning System only neB #e8101)

M13KE is a simple M13mp19 derivative into which cloning sites have been introduced at the 5′ end of gene III for display of short peptide sequences as N-terminal pIII fusions . The sequence of M13KE is available at neb .com; see DNA Maps and Sequences . Because this is a phage, rather than a phagemid vector, all 5 copies of pIII on the surface of each virion will be fused to the cloned peptide . Displayed proteins longer than 30–50 amino acids may have a deleterious effect on the infectivity function of pIII, therefore this vector is suitable only for the dis-play of peptides and small proteins (unpublished observations) . The small insert size does not appreciably attenuate phage replication, allowing the vector to be propagated as phage, rather than as a plasmid (i .e ., titer for plaques, not colo-nies) . Thus the vector carries neither a plasmid replicon nor antibiotic resistance . This simplifies the intermediate amplification steps during panning considerably, as it is not necessary to express antibiotic genes before plating or to use helper

phage during amplification . The steps necessary to clone a peptide library into

M13KE are outlined below . To clone a single peptide sequence, reactions can be scaled down appropriately .

Preparation of electrocompetent Cells

This procedure will generate a sufficient quantity of electrocompetent cells for test ligations and large-scale library production. The use of commercially available competent cells on this scale is financially prohibitive. In this protocol, 10% glycerol may be used in all washes for improved efficiency.

1 . Inoculate 6 liters of LB medium (in six 4-liter Erlenmeyer or

2 .8-liter Fern-

bach flasks to maximize aeration) with 1/100 volume (10 ml per liter) of an overnight culture of ER2738 that has been grown at 37°C with shaking . The overnight should be inoculated with ER2738 from a fresh LB/Tet plate (1–3 days) .

2 . Incubate the cultures at 37°C with vigorous shaking (> 250 rpm) until cul-

tures reach an OD

600

of 0 .5–1 .0 .

3 . Chill flasks on ice for 30 minutes, and harvest the cells by centrifugation at

5000 g for 15 minutes at 4°C . Discard the supernatant .

4 . Suspend each pellet in 1 liter of ice-cold autoclaved H

2O (Milli-Q? or equiva-

lent) . Centrifuge as before and discard the supernatant .

5 . Suspend each pellet in 0 .5 liter of ice-cold autoclaved H

2O . Combine in 3

bottles and centrifuge as before . Discard supernatant .

6 . Suspend the pellets in 120 ml total of ice-cold autoclaved 10% (v/v) glycerol

in water . Combine in a single bottle and centrifuge at 8000 g for 10 minutes

at 4°C . Discard as much of the supernatant as possible without disturbing the

pellet .

7 . Suspend the pellet in 12 ml of ice-cold 10% glycerol . Dispense into 100-μl

aliquots and immediately freeze in dry ice-ethanol bath . Store at –80°C .

8 . Check electrocompetence by electroporating 1 ng of M13 RF DNA (e .g .,

M13KE or M13mp19, diluted in a low ionic strength buffer such as TE) into a

freshly thawed aliquot of electrocompetent cells, according to the manufac-

turer’s instructions . Suggested parameters (Bio-Rad Gene-Pulser) are: 25 μF,

200 Ω, 2 .5 kV .

9 . Immediately add 1 ml of SOC medium and incubate at 37°C for 30–45

minutes . This gives the cells sufficient time to recover without allowing phage

production and infection of untransformed cells, which could result in inflated

electroporation efficiencies .

10 . Prepare 102-, 103-, and 104-fold dilutions of the outgrowth in LB . Transfer

10 μl of each dilution to a test tube containing 3 ml of Top Agar and 200 μl of

a mid-log culture of ER2738, equilibrated at 45°C . Vortex briefly and spread

on LB/IPTG/Xgal plates . Incubate overnight at 37°C and count blue plaques

the next day .

11 . Electroporation efficiencies should be at least 1 x 109 transformants/μg .

10

11The following procedure (adapted from references 5 and 7) is specific for the M13

cloning vector M13KE, but could easily be adapted for other phage (but NOT phagemid) vectors. More details can be found in reference 49.

1 . Design a library oligonucleotide following the convention in Figure 4 . Bear in mind that the sequence VPFYSHS preceding the leader peptidase cleavage site is part of the pIII signal sequence and should not be altered . The first residue of the displayed peptide will immediately follow this se-quence . For randomized positions, relative representations of each amino acid can be improved by limiting the third position of each codon to G or T (= A or C on the synthetic library oligonucleotide) . We recommend including a short spacer sequence between the randomized segment and the first native pIII residue to improve target accessibility to the displayed peptide, e .g . the spacer Gly-Gly-Gly between the random peptide and the Ser-Ala-Glu (SAE) shown in Figure 4 . This sequence can also include a protease cleavage site to allow elution of bound phage by protease diges-tion (50, 51) . Pentavalency of the displayed peptide does not prevent

...AACGTGAAAAAATTATTATTCGCAATTCCTTTAGTGGTACCTTTCTATTCTCACTCGGCCGAA... ...TTGCACTTTTTTAATAATAAGCGTTAAGGAAATCACCATGGAAAGATAAGAGTGAGCCGGCTT... M K K L L F A I P L V V P F Y S H S A E ...gene III Acc65 I Kpn I Eag I

M13KE:Mature pIII

Leader Peptidase Leader Sequence

5′ CATGCCCGGGTACCTTTCTATTCTC 3′3′ GGGCCCATGGAAAGATAAGAGTGAGA(NNN)n AGCCGGCTTTGTAC 5′Library Oligonucleotide Extension Primer

Klenow, dNTP’s

5′ CATGCCCGGGTACCTTTCTATTCTCACTCT(NNN)n TCGGCCGAAACATG 3′3′ GTACGGGCCCATGGAAAGATAAGAGTGAGA(NNN)n AGCCGGCTTTGTAC 5′

Acc65 I Kpn I Eag I

Acc65 I, Eag I

5′ GTACCTTTCTATTCTCACTCT(NNN)n TC 3′3′ GAAAGATAAGAGTGAGA(NNN)n AGCCGG 5′

...AACGTGAAAAAATTATTATTCGCAATTCCTTTAGTGGTACCTTTCTATTCTCACTCT(NNN)n TCGGCCGAA... ...TTGCACTTTTTTAATAATAAGCGTTAAGGAAATCACCATGGAAAGATAAGAGTGAGA(NNN)n AGCCGGCTT... M K K L L F A I P L V V P F Y S H S X n S A E ...Acc65 I Kpn I Eag I

Leader Sequence

12

protease release of bound phage (unpublished data) . The oligonucleotide should be synthesized on a minimum of 0 .2 μmol scale, gel-purified (48), and accurately quantitated by measuring the OD 260 in a spectrophotometer (1 absorbance unit at 260 nm = 20 μg/ml of single stranded DNA) .

2 . Anneal 5 μg of the library oligonucleotide with

3 molar equivalents of the universal extension primer 5′-HO CATGCCCGGGTACCTTTCTATTCTC-3′ (approximately

4 μg for a 90-nucleotide library oligonucleotide) in a total volume of 50 μl TE containing 100 mM NaCl . Heat to 95°C and cool slowly (15–30 minutes) to less than 37°C in a thermal cycler or water bath .3 . Extend the annealed duplex as follows (mix in the given order)

H 2O 119 μl 10X NEBuffer 2 20 annealed duplex 50 10 mM dNTPs 8 Klenow fragment (NEB #M0210) (5 Units/μl ) 3

200 μl

Incubate at 37°C for 10 minutes, then 65°C for 15 minutes . Save 4 μl for later analysis (Step 5) .4 . The extended duplex is then cut using EagI and either, KpnI or Acc65I . We favor digestion as follows:

Extension reaction 196 μl H 2O 149 BSA (10 mg/ml) 4 10X NEBuffer 2 40 EagI-HF (10 Units/μl ) 5 KpnI (10 Units/μl ) 6

400 μl

Extension reaction 196 μl H 2O 154 10X NEBuffer 4 40 EagI-HF (10 Units/μl ) 5 KpnI-HF (10 Units/μl ) 5

400 μl

Incubate at 37°C for 3–5 hours .* Purify the DNA by phenol/chloroform extraction, chloroform extraction and ethanol precipitation . *EagI-HF is not recommended for > 1 hour digestions .

5 . Gel-purify the digested duplex on an 8% nondenaturing polyacrylamide gel (48), including as molecular weight markers, 4 μl of the undigested duplex as well as Low Molecular Weight DNA Ladder (NEB #N3233) . Visualize by ethidium bromide staining, and excise the digested duplex from the gel, minimizing UV exposure time . Mince the excised band and elute the DNA by shaking overnight in several volumes of 100 mM sodium acetate, pH 4 .5, 1 mM EDTA, 0 .1% SDS at 37°C .

6 . Briefly microfuge to separate the gel fragments from the elution buffer,

and transfer the supernatant to a clean tube . Repeat wash to improve

yield, if desired . Purify the DNA duplex from the supernatant by phenol/

chloroform extraction, chloroform extraction and ethanol precipitation

(48) . Resuspend the pellet in 50 μl of TE and quantitate a small amount

by PAGE or spectrophotometrically . One μg of purified insert is more than

sufficient for a library of complexity 109 .

7 . For a high-complexity library, digest 10–20 μg of M13KE vector with 10

units per μg of EagI-HF and 15 units per μg KpnI or enzymes of choice, for every 40 μl of 1X NEBuffer (total volume = 400–800 μl) . Gel purify using

standard methods (β-Agarase, QIAGEN?, etc) . Quantitate a small amount

of purified cut vector on an agarose gel or spectrophotometrically .

8 . Optimize the ligation conditions . Suggested starting parameters per 20 μl

ligation: 40 and 100 ng of cut vector; 3:1, 5:1 and 10:1 molar excess of

cut duplex; 2 μl of 10X ligase buffer; and 200 units (= 3 Weiss units) of T4 DNA Ligase (NEB #M0202) . Incubate overnight at 16°C .

9 . Heat-kill the test ligations at 65°C for 15 minutes, then electroporate 1 μl

of each into 100 μl of electrocompetent ER2738 or other F+ strain (for

suggested electroporation parameters see step 8 in the previous section) .

Outgrowths are carried out in 1 ml of SOC medium for 30–45 minutes at

37°C with shaking .

10 . Prepare 10, 100, and 1000-fold dilutions of the outgrowth in LB . Transfer

10 μl of each dilution to a test tube containing 3 ml of top agar + 200 μl

of a mid-log culture of ER2738, equilibrated at 45°C . Vortex briefly and

spread on LB/IPTG/Xgal plates . Incubate overnight at 37°C and count blue plaques the next day .

11 . Scale up the protocol using the highest plaque/μg ratio to desired library

complexity . For example, a library with a compexity of 1 x 109 clones

would require a 5 μg ligation if the test ligations yield a ratio of 2 x 108

plaques/μg of vector . Use no more than 500 μl per individual ligation reac-

tion; use multiple tubes if necessary .

12 . Purify the large-scale ligation by phenol/chloroform extraction, chloroform

extraction and ethanol precipitation . Wash with 70% ethanol to desalt . Re-

suspend the DNA in low salt buffer and electroporate as described above .

To reduce the likelihood of cells picking up more than one DNA sequence,

the ligation should be divided and electroporated using as many cuvettes

as convenient . For a 10–20 μg scale ligation we typically carry out 100

electroporations, using 3 μl of resuspended ligated DNA per 100 μl of

electrocompetent cells .

13 . Add 1 ml of SOC to each cuvette immediately after electroporation . For

high-complexity libraries it may be convenient to pool the SOC outgrowths in groups of 5 . Each outgrowth (or pool of 5) should be incubated for

30–45 minutes (no longer) before amplification . Titer several outgrowths

or pools (as in Step 10) prior to amplification in order to obtain library

complexity .

13

14

14 . Amplify the electroporated cells by adding 20 ml of pooled SOC outgrowths to 1 liter of early-log cells (OD 600 0 .01–0 .05) in LB medium . Incubate with vigorous aeration (250 rpm) at 37°C for 4 .5 to 5 hours . Centrifuge at 5000 g for 20 minutes at 4°C . Transfer the supernatant to a clean bottle and discard the cells .

15 . Recover the phage from the supernatant by adding 1/6 volume of 20% PEG/2 .5 M NaCl and incubating overnight at 4°C . Pellet the phage by cen-trifugation at 5000 g for 20 minutes at 4°C . Discard the supernatant .

16 . Thoroughly resuspend the phage pellet in 100 ml of TBS by gently rocking over ~1–3 hour or overnight at 4°C . Remove residual cells by centrifugation at 5000 g for 10 minutes at 4°C .

17 . Transfer the supernatant to a new tube and discard the pellet . Reprecipitate the phage by adding 1/6 volume of 20% PEG/2 .5 M NaCl and incubating for 1 hour at 4°C . Centrifuge at 5000 g for 20 minutes and discard the superna-tant .

18 . Resuspend the final library in 10–40 ml of TBS by gentle rocking for 24–48 hours at 4°C . For long-term storage, add an equal volume of sterile glycerol, mix thoroughly and store at –20°C . The titer of the library should remain constant for several years at this temperature . Further amplification of the library is not recommended, as sequence biases may occur upon reamplification .

Surface Panning Procedure (Direct target Coating)The most straightforward method of affinity partitioning (panning) involves directly coating a plastic surface with the target of interest (by nonspecific hydrophobic and electrostatic interaction), washing away the excess, and passing the pool of phage over the target-coated surface (Figure 1). Because of its relative simplicity, we recommend trying the direct coat-ing method described below first. If no clear consensus binding sequence emerges, then proceed with either of the solution binding procedures.

Day one

Depending on the available quantity of target molecule and the number of different targets being panned against simultaneously, panning can be carried out in individual sterile poly-styrene petri dishes, 12- or 24-well plates, or 96-well microtiter plates. Coat a minimum of 1 plate (or individual well) per target. It is not productive to do a separate negative control panning experiment without target. Volumes given in the following procedure are for 60 x 15 mm petri dishes, with volumes for microtiter wells given in parentheses. For wells of intermediate size adjust volumes accordingly, but in all cases the number of input phage should remain the same (1011 virions).

1 . Prepare a solution of 10–100 μg/ml of the target in 0 .1 M NaHCO 3, pH 8 .6 . Alternative buffers (containing metal ions etc .) of similarly high ionic strength (e .g ., TBS) can be used if necessary for stabilizing the target mol-ecule . Do not add DTT if using the Ph .D .-C7C library .

15

2 . Add 1 .5 ml of this solution (150 μl if using microtiter wells) to each plate (or well) and swirl repeatedly until the surface is completely wet (this may take some effort as the solution may bead up) .

3 . To coat plates with target, incubate overnight at 4°C with gentle agitation in a humidified container (e .g ., a sealable plastic box lined with damp paper towels) . Store plates at 4°C in humidified container until needed . Plates coated with antibodies or streptavidin can usually be stored for several weeks (depending on target stability); discard if mold is evident on the paper towels .

Day two 4 . Inoculate 10 ml of LB+Tet medium with ER2738 . This culture will be used for titering in Step 11 and can be used in 5–10 hours . If amplifying the eluted phage on the same day (see step 12 and note), also inoculate 20 ml of LB medium in a 250-ml Erlenmeyer flask (do not use a 50-ml conical tube) with ER2738 . Incubate both cultures at 37°C with vigorous shaking . Incubate the titering culture until needed; carefully, monitor the 20-ml culture so that it does not grow beyond early-log phase (OD 600 0 .01–0 .05), for use in Step 12 .

5 . Pour off the coating solution from each plate and firmly slap it face down onto a clean paper towel to remove residual solution . Fill each plate or well completely with blocking buffer . Incubate for at least 1 hour at 4°C .

6 . Discard the blocking solution as in step 5 . Wash each plate rapidly 6 times with TBST (TBS + 0 .1% [v/v] Tween-20) . Additional components (e .g ., metal ions) can be added to the TBST if necessary to stabilize the target molecule (do not add DTT if using the Ph .D .-C7C library) . Coat the bottom and sides of the plate or well by swirling, pour off the solution, and slap the plate face down on a clean paper towel each time . (If using a 96-well microtiter plate, an automatic plate washer may be used .) Work quickly to avoid drying out the plates .

7 .

Dilute a 100-fold representation of the library (e .g ., 2 x 1011 phage for a library with 2 x 109 clones) with 1 ml of TBST (100 μl if using microtiter wells) . Pipette onto coated plate and rock gently for 10–60 minutes at room temperature . (See Appendix for discussion of binding time consider-ation, page 29) 8 . Discard nonbinding phage by pouring off and slapping plate face-down onto a clean paper towel .

9 . Wash plates 10 times with TBST as in step 6 . Use a clean section of paper towel each time to prevent cross-contamination .

10 . Elute bound phage with 1 ml (100 μl if using microtiter wells) of an ap-propriate elution buffer for the interaction being studied . Typically this will be a solution of a known ligand for the target (0 .1–1 mM) in TBS, or a solution of the free target (~100 μg/ml in TBS) to compete the bound phage away from the immobilized target on the plate . Rock the elution

mixture gently for 10–60 minutes at room temperature . Pipette eluate

into a microcentrifuge tube . Alternatively, a general buffer for nonspecific

disruption of binding interactions is 0 .2 M Glycine-HCl (pH 2 .2), 1 mg/

ml BSA . If using this buffer, rock gently for no more than 10–20 minutes,

pipette eluate into a microcentrifuge tube, and neutralize with 150 μl (15

μl for microtiter wells) of 1 M Tris-HCl, pH 9 .1 .

11 . Titer a small amount (~1 μl) of the eluate as described in General M13

Methods (page 8) . Plaques from the first or second round eluate titering

can be sequenced if desired (page 22) .

12 . Amplify the rest of the eluate by adding the eluate to the 20-ml ER2738

culture from Step 4 (should be early-log at this point) and incubating with

vigorous shaking for 4 .5 hours at 37°C .

Note: The remaining eluate can be stored overnight at 4°C at this point, if preferred, and amplified the next day. In this case, inoculate 10 ml of LB+Tet with ER2738 and incubate with shaking overnight at 37°C. The next day, dilute the overnight culture 1:100 in 20 ml of LB in a 250-ml Erlenmeyer flask (do not use a 50 ml conical tube) and add the unamplified eluate. Incubate with vigorous shaking for 4.5 hours at 37°C and proceed to Step 13.

13 . Transfer the culture to a centrifuge tube and spin for 10 minutes at

12,000 g at 4°C . Transfer the supernatant to a fresh tube and re-spin

(discard the pellet) .

14 . Transfer the upper 80% of the supernatant to a fresh tube and add to it

1/6 volume of 20% PEG/2 .5 M NaCl . Allow the phage to precipitate at 4°C

for at least 2 hours, preferably overnight .

Day three

15 . Spin the PEG precipitation at 12,000 g for 15 minutes at 4°C . Decant and

discard the supernatant, re-spin the tube briefly, and remove residual

supernatant with a pipette . The phage pellet should be a white finger print

sized smear on the side of the tube .

16 . Suspend the pellet in 1 ml of TBS . Transfer the suspension to a micro-

centrifuge tube and spin at maximum (14,000 rpm) for 5 minutes at 4°C

to pellet residual cells .

17 . Transfer the supernatant to a fresh microcentrifuge tube and reprecipitate

by adding 1/6 volume of 20% PEG/2 .5 M NaCl . Incubate on ice for 15–60

minutes . Microcentrifuge at 14,000 rpm for 10 minutes at 4°C, discard

the supernatant, re-spin briefly, and remove residual supernatant with a

micropipet .

18 . Suspend the pellet in 200 μl of TBS . Microcentrifuge for 1 minute to pel-

let any remaining insoluble material . Transfer the supernatant to a fresh

tube . This is the amplified eluate .

16

19 . Titer the amplified eluate as described in General M13 Methods (page

8) on LB/IPTG/Xgal plates . The eluate can be stored for up to 3 weeks at

4°C . For long-term storage, add an equal volume of sterile glycerol and

store at –20° C .

20 . Coat a plate or well for the second round of panning as in Steps 1–3

above

Days Four and Five

21 . Count blue plaques from the titering plates in Step 19 and determine the

phage titer, which should be on the order of 1013–14 pfu/ml . Use this value

to calculate an input volume corresponding to the input titer in Step 7 . If

the phage titer of the amplified eluate is too low, succeeding rounds of

panning can be carried out with as little as 109 pfu of input phage .

22 . Carry out a second round of panning: repeat steps 4–18 using the

calculated amount of the first round amplified eluate as input phage, and

raising the Tween concentration in the wash steps to 0 .5% (v/v) .

23 . Titer the resulting second round amplified eluate on LB/IPTG/Xgal plates .

24 . Coat a plate or well for the third round of panning as in Steps 1–3 above . Day Six

25 . Carry out a third round of panning: repeat steps 4–10, using the second

round amplified eluate at an input titer equivalent to what was used in the

first round (step 7), again using 0 .5% Tween in the wash steps .

26 . Titer the unamplified third round eluate as in step 11 on LB/IPTG/Xgal

plates . It is not necessary to amplify the third round eluate . Plaques from

this titering can be used for sequencing: time the procedure so that plates are incubated at 37°C for no longer than 18 hours, as deletions may

occur if plates are grown longer . Stored at 4°C, plates should be used

to pick plaques within 1–3 days of plating . The remaining eluate can be

stored at 4°C for at least one week .

27 . For preparation of individual clones for sequencing or ELISA, set up a 10-

ml overnight culture of ER2738 from a colony, not by diluting the titering

culture . Proceed with plaque amplification, page 22 . Do not amplify the

third round eluate and carry out a fourth round of panning unless the

characterization shows no clear consensus sequence or phage ELISA

results are negative .

Control Panning experiment with Streptavidin target

Follow the above procedure using streptavidin as the target, including 0 .1 μg/

ml streptavidin in the blocking solution to complex any biotin or biotinylated protein in the BSA . Elute bound phage with 0 .1 mM biotin in TBS for at least

30 minutes . After 3 rounds of enrichment/amplification, the consensus se-quence for streptavidin-binding peptides should include the motif His-Pro-Gln (HPQ) (7) .

17

18

Solution-phase Panning with a Biotinylated target and Streptavidin Plate Capture

As an alternative to directly coating a plate with the target molecule, the target can be reacted with the phage in solution, followed by affinity capture of the phage-target complexes (6). Depending on the target, direct coating can result in an inaccessible ligand binding site, either due to steric blocking or partial denaturation of the target along the surface. Affinity capture requires some sort of affinity tag on the target; one way this can be accomplished is by biotinylating the target and capturing the complexes with a streptavidin-coated polystyrene plate. Alternatively, the biotinylated target–phage com-plexes can be captured with streptavidin-agarose beads, using the general bead selection procedure described on page 19. Capture of the target can be accomplished either before or after addition of phage.

Biotinylation of target

1 . Dissolve or dilute

2 mg of target protein in 1 ml of 50 mM NaHCO 3, pH 8 .5 . Other buffers may be used if necessary to maintain stability of target, but do NOT use buffers with free primary or secondary amine groups (e .g ., Tris, ethanolamine) .

2 . Immediately prior to use, dissolve ~1 mg of sulfo-NHS-LC-Biotin (avail-able from Pierce, cat . #21335) in 1 ml of H 2O . Vortex vigorously . Add 74 μl of this (18 mM) solution to the target solution . This reagent is a water-soluble activated ester of biotin that specifically targets the ε-amine of solvent-accessible (surface) lysine residues . The remaining solution should be discarded, as the ester hydrolyzes rapidly upon storage .

3 . Incubate the biotin–target mixture for 2 hours on ice .

4 . Remove unreacted biotin by dialysis against a minimum of 2 one-liter changes of TBS, gel-filtration, or ultrafiltration . Alternate buffers of simi-lar ionic strength can be used at this point .

5 .

Quantitate the biotinylated protein by Bradford or Lowry assay . Bioti-nylation can be confirmed by Western blot detection or ELISA using commercially available anti-biotin antibodies . The degree of biotinylation can be quantitated by a colorimetric assay based on displacement of the dye HABA (2-[4′-Hydroxyazobenzene]-benzoic acid), also available from Pierce (cat . #28010) . Based on results obtained at NEB, the described reaction conditions result in an average of 2 biotinylated lysines per ap-proximately 25-kDa protein molecule .Panning with Surface Streptavidin Capture

Follow the panning procedure described above for the surface panning, direct target coating method (page 14), but coat the plates with streptavidin (100 μg/ml of streptavidin in 0.1 M NaHCO 3, pH 8.6) instead of target. The blocking buffer should contain 0.1 μg/ml streptavidin in order to complex any biotin or biotinylated protein in the BSA. Replace Step 7 in the direct target coating method (page 15) with the following:

噬菌体展示肽库技术的研究及应用进展

2010.01

2010.01 的目的基因克隆进表达载体(fuse phage),与噬菌体的外壳蛋白基因融合表达,使得一个噬菌体上含有一种序列的肽。与有机合成法相比较,该方法有其独特的优越性:它可将特定分子的基因型和其表型统一在同一个病毒颗粒内,并且将选择能力和扩增能力联系在一起,即通过与配体的结合从数量众多的多样性群体中选择出表达有相应配体的噬菌体颗粒,再通过感染大肠杆菌使选择出的噬菌体颗粒得到扩增。其不足在于肽库的容量及短肽的大小受到了限制,且只能表达L 型天然氨基酸形成的短肽。 3噬菌体肽库的筛选技术 如何从噬菌体肽库中筛选到特异的重组噬菌 体是肽库技术的关键。经典的方法有两种:①将纯抗体包被在固相介质上,如酶标板、免疫试管或亲和层析柱,然后加入待筛选的噬菌体,洗去非亲和性的噬菌体,回收高亲和性的噬菌体;②将抗体与生物素基因相连,再将其固定在包被有 Streptavidin 的磁珠上对噬菌体进行筛选。3.1 宿主菌直接洗脱 经典筛选过程中,一般利用pH 的变化来洗脱与目标分子结合的噬菌体,这可能对噬菌体造成伤害。利用噬菌体与宿主之间的亲和性可以直接用宿主菌来洗脱,以避免对筛选的影响,并且将洗脱和感染合并到一步[7]。 3.2双层膜筛选系统 获得重组噬菌体蛋白的方法是将筛选出的特 异噬菌体转入不含校正基因的菌株,将外源蛋白以可溶性蛋白的形式表达出来。通过细胞膜间隙最终进入培养基质中。针对这种情况,Skerra 等[8]建立了双层膜筛选系统。第一层膜为亲水性多孔膜,这种膜对蛋白质的结合能力小,孔径能让蛋白质分子自由通过,但细胞不能通过;第二层膜为疏水膜,膜表面包被目标蛋白(抗原或抗体),两种膜分别覆盖在固体培养基上。细胞在第一层膜上培养一段时间后,将这层膜转移到第二层膜上培养,分泌的可溶性蛋白透过第一层膜,与第二层膜上的目标蛋白结合,然后再用已知的抗原或抗体筛选。这种方法避免了蛋白常遇到的细胞碎片的干扰,提高了筛选 效率。 3.3选择性感染噬菌体筛选系统 传统的筛选系统中基因重组的噬菌体仍有野 生型的gene Ⅲ蛋白的存在,因重组噬菌体具有感染能力,这导致在筛选后扩增时特异性和非特异性的噬菌体均可以进入宿主菌增殖。选择性感染噬菌体筛选系统把筛选和扩增合并为一步,只有特异性的噬菌体才能感染宿主菌扩增,而非特异性噬菌体却不能。此筛选系统是以噬菌体感染宿主菌的机理为基础,其基本原理是把其中之一用特定的多肽或蛋白替代,使噬菌体丧失感染力,再通过这些多肽与其配基的结合恢复感染能力。这样只有特异性的噬菌体可以通过配基配体结合才能恢复感染能力,进入宿主菌细胞内繁殖[9]。具体的做法有两种,其一是将噬菌体所有P Ⅲ外壳蛋白的N 端用所展示的多肽或蛋白取代,使其丧失感染能力,再把与所筛选的目的多肽或蛋白的特异配基同P Ⅲ蛋白的N 端相连作为筛选的探针,只有特异 性噬菌体才能与连接有P Ⅲ蛋白N 端的配基相结合,这种结合使噬菌体重新具有P Ⅲ蛋白的N 端,因而具有感染力;其二是将作为配基的多肽或蛋白融合在E.coli 的纤毛上,这样纤毛失去了介导的能力,只有特异性的噬菌体可与修饰了的纤毛结合,从而恢复纤毛的介导能力,使特异性的噬菌体进入细胞体内增殖,这一系统显示了较高的筛选效率。 4噬菌体肽库的应用4.1 抗原表位的研究 传统的抗原表位分析主要是通过分析抗原经 物理化学降解得到的片段或人工合成一系列来源于抗原的肽段与相关抗体的结合活性而得到,此方法成本高,必须首先确定蛋白质的氨基酸序列,才能合成相应的肽,而在实际研究中,许多蛋白质的序列并不清楚。噬菌体肽库技术将抗体作为受体暴露于肽库中,筛选到能与特异性抗体相结合的重组噬菌体肽的序列,弥补了合成肽筛选的不足。在抗原-抗体的识别研究中,不必源于天然抗原,可以从随机肽库中直接筛选出能和抗体结合的表位。尽管有时与天然表位在氨基酸顺序上可能不一致,但是

噬菌体展示肽库的筛选方法及其应用

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噬菌体抗体库技术 张爱华 余模松 【摘要】 噬菌体抗体库技术是20世纪90年代继噬菌体展示技术发展而来。这一技术彻底改变了抗体制备的传统途径,使抗体工程技术进入了一个新的发展阶段。噬菌体抗体库技术是迄今发展最成熟、应用最广泛的抗体库技术。本文对与基因工程抗体相关的噬菌体展示技术以及噬菌体抗体库技术作一综述。 【关键词】 噬菌体展示技术;噬菌体抗体库技术;免疫抗体库 【中图分类号】R 392.11 【文献标识码】A 【文章编号】1673-4211(2006)01-0013-04 作者单位:430060武汉生物制品研究所免疫学研究室 20世纪80年代,随着DNA 重组技术的进展和抗体基因结构的阐明,产生了基因工程抗体技术。早期用于构建基因工程抗体的抗体基因主要来源于小鼠杂交瘤细胞。由于要获得杂交瘤细胞必须经过动物免疫、细胞融合及克隆筛选这样一个长期、复杂的过程;而且利用杂交瘤技术很难制备人源抗体和抗自身抗原或弱免疫原性抗原的抗体,所以限制了基因工程抗体技术的推广和应用。20世纪90年代,人们又将噬菌体展示技术应用到抗体的表达和克隆,将组建亿万种不同特异性抗体可变区基因文库和抗体在大肠杆菌功能性表达与高效快速的筛选手段结合起来,产生了噬菌体抗体库技术,这一技术彻底改变了抗体制备的传统途径,由此抗体工程技术进入了一个新的发展阶段。噬菌体抗体库技术是迄今发展最成熟、应用最广泛的抗体库技术[1]。 1 噬菌体展示技术 1985年Smith [2]首次阐述了噬菌体展示技术,该技术通过将外源蛋白或肽段的基因克隆到丝状噬菌体的基因组DNA 中,与噬菌体的外壳蛋白形成融合蛋白,从而使该外源分子呈现于噬菌体表面。该技术的主要特点是将特定分子的基因型和表型统一在同一噬菌体颗粒内,即在噬菌体表面表达特定蛋白,而在噬菌体核心DNA 中含有该蛋白的结构基因,通过表型筛选就可以获得其编码基因,因此噬菌体展示技术是一种强有力的基因表达筛选技术。 蛋白质可以展示在更小的丝状噬菌体颗粒的表面,这就是噬粒展示系统[3] 。噬粒的基因组中包含丝状噬菌体的基因间隔区,包括病毒颗粒和互补链合成的复制起始点以及发夹包装信号,同时也含 有质粒的复制起始点和抗性基因。噬粒也可以像质粒一样操作,假如需要,可以直接在细菌中表达目的蛋白。采用丝状辅助噬菌体感染细菌可以激活噬菌体的复制起始点,从而导致单链噬粒DNA 被包装进由辅助噬菌体蛋白形成的丝状噬菌体样的颗粒中,由于辅助噬菌体缺乏包装信号,所以产生的大多数噬菌体为含有噬粒单链DNA 的颗粒[4],将这种噬粒和噬菌体混合物感染细菌,筛选具有抗性的克隆,这种带抗性的克隆只含有噬粒DNA,可再次通过辅助噬菌体的感染进行扩增。由于噬粒颗粒可以传播抗性基因,所以有时又把这种颗粒称为“转导颗粒”。 p Ⅲ蛋白是一种最常用于展示外源片段的噬菌体外壳蛋白。其缺点是每个噬菌体颗粒表面仅有5个p Ⅲ分子,但是它的优点是带大的插入片段的p Ⅲ分子可以被很好地包装进噬菌体颗粒中。在绝大多数情况下,外源蛋白插入在信号序列和p Ⅲ蛋白第一结构域(N 1)的起始位点之间,一方面外源蛋白不影响噬菌体的包装,另一方面,外源蛋白位于包装颗粒的最末端,所以当通过p Ⅳ蛋白孔时,其空间位阻较小。但问题是大片段的插入降低了噬菌体的感染能力,甚至使噬菌体无感染性,因此限制了某些特殊蛋白的选择。这一问题可以通过杂交噬菌体得到解决,即仅有一个p Ⅲ分子用于展示蛋白,具体方案是将外源蛋白构建到噬粒载体中,转化细菌后,再用辅助噬菌体超感染,这样细菌中的大多数p Ⅲ蛋白为来源于辅助噬菌体的野生型分子[5] 。噬菌体展示活性蛋白的能力主要依赖于以下几个方面的因素,比如融合蛋白能否被正确地转移到细菌内膜,能否正确地折叠,能否在外周质中逃避降解以及能否被包装进噬菌体颗粒中。外源蛋白也可以插入到p Ⅲ蛋白的N1和N2以及N 2和CT 结 ? 13?国际生物制品学杂志2006年2月第29卷第1期 Int J Biolog icals,February 2006,Vol 29,No.1

噬菌体展示技术解析

噬菌体展示技术解析 噬菌体展示技术经过近20年的发展和完善,已被广泛应用于抗原抗体库的建立、药物设计、疫苗研究、病原检测、基因治疗、抗原表位研究及细胞信号转导研究等。噬菌体展示系统模拟了自然免疫系统,使我们有可能模拟体内抗体生成过程,构建高亲和力抗体库。由于噬菌体展示技术实现了基因型和表型的有效转换,使研究者在基因分子克隆基础上实现了蛋白质构象体外控制,从而为获取具有良好生物学活性的表达产物提供了强有力手段。另外,噬菌体展示技术已成为不经过免疫获取特异性人源抗体的新途径,为获取对人类和动物疾病有诊断和治疗价值的单克隆抗体提供了重要手段。 应用面这么高大上,那么原理到底是什么呢?那接下来将由小编为您揭开其神秘的面纱! 一、噬菌体展示技术的原理 噬菌体展示技术(phage display)是将多肽或蛋白质的编码基因插入噬菌体外壳蛋白结构基因的适当位置,在阅读框正确且不影响其他外壳蛋白正常功能的情况下,使外源多肽或蛋白与外壳蛋白融合表达,融合蛋白随子代噬菌体的重新组装而展示在噬菌体表面。展示到噬菌体表面的多肽或蛋白保持相对独立的空间结构和生物活性,可以与靶分子结合和识别。噬菌体展示的肽库或蛋白库与固相抗原结合,洗去未结合的噬菌体,然后用酸碱或者竞争的分子洗脱下结合的噬菌体,中和后的噬菌体感染大肠杆菌扩增,经过3-5轮的富集,逐步提高可以特异性识别靶分子的噬菌体比例,最终获得识别靶分子的多肽或者蛋白。下图粗略展示了技术筛选的过程: 二、噬菌体展示系统的分类 1.M13噬菌体展示系统 M13噬菌体属于单链环状DNA病毒,其基因组为6.4kb,编码10种蛋白,其中5种为结构蛋白,包括主要衣壳蛋白的PⅧ和次要衣壳的pⅢ、pⅥ、PⅦ和PⅨ。其中,pⅢ和PⅧ是噬菌体展示中最常用的两种蛋白,构建了pⅢ和PⅧ展示系统。pⅢ蛋白分子量为42 kDa,

噬菌体展示技术操作步骤

筛选多肽 试剂及配制 (1)LB培养基: 胰蛋白胨10g 酵母提取物5g NaCl 5g 溶于去离子水,至1L,高压灭菌,4℃保存。 (2)IPTG/Xgal: 称取1.25g IPTG,1.0g Xgal,溶于二甲基甲酰胺,至总体积25ml,-20℃避光保存。(3)LB/IPTG/Xgal平板: 1L LB培养基+15g琼脂粉,高压灭菌,冷却至70℃以下,加入1ml IPTG/Xgal,立即铺板,4℃避光保存。 (4)顶层琼脂糖凝胶: 胰蛋白胨10g 酵母提取物5g NaCl 5g MgCl2.6H2O 1g 琼脂糖7g 溶于适量去离子水中,至总体积为1L。高压灭菌,分装为50ml/份,室温保存,使用时于微波炉内融化。 (5)2×M9盐: Na2HPO412g KH2PO46g NaCl 1g NH4Cl 2g 溶于适量去离子水中,至终体积为1L。 (6)小型平板: 500ml 2×M9盐 500ml 3%琼脂粉 20ml 20%葡萄糖 2ml 1M MgSO4 0.1ml 1M CaCl2 1ml 硫胺素(10mg/ml) 在混合之前,将上述成分分别高压灭菌并冷却至70℃以下,葡萄糖和硫胺素采用过滤除菌。平板储存于4℃。 (7)封闭缓冲液: 0.1M NaHCO3(PH 8.6) 5mg/ml BSA 0.02% NaN3 过滤除菌,储存于4℃。 (8)TBS: 50mM Tris-HCl (PH 7.5) 150mM NaCl

高压灭菌,室温储存。 (9)PEG/NaCl: 20%(w/v)聚乙二醇-8000 2.5M NaCl 高压灭菌,室温储存。 (10)碘化物缓冲液: 10mM Tris-HCl (PH 8.0) 1mM EDTA 4M NaI 室温避光保存。 操作步骤: 1.第一天 (1)以0.1M NaHCO3(PH 8.6)制备 100μg/ml的靶分子溶液。如果需要稳定靶分子,可以用含有金属离子的相似离子强度缓冲液。 (2)在每孔内加入1.5ml 靶分子溶液,重复涡旋直至表面完全湿润(这一步要多注意,尽量避免溶液形成液珠)。 (3)在湿盒中,4℃温和振荡孵育过夜。4℃保存于湿盒中备用。 2.第二天 (4)将ER2537(滴度测定时用来铺板的细菌培养物)接种至10ml LB培养基中。如果是在同一天扩增洗脱的噬菌体,也可以将ER2537过夜培养物接种于含有20ml LB培养基的250ml锥形瓶中(比例为1:100)。37℃剧烈振摇。 (5)将平皿反扣在洁净的纸巾上,去除包被液,加满封闭液,4℃孵育至少1h。 (6)去除包被液。用TBST(TBS+0.1%Tween-20)快速洗涤6次。反复旋转确保孔底及孔侧面都被洗涤。按照步骤5的方法去除洗涤液(也可利用自动洗板机洗涤)。洗涤要迅速,避免平皿干燥。(注意每次更换纸巾,以避免交叉污染)。 (7)用1ml TBST稀释4×1010噬菌体(10μl原库),加至包被后的平皿内,室温下轻缓摇动10-60min。 (8)以步骤5的方法去除未结合的噬菌体。 (9)以步骤6的方法用TBST洗涤板子10次,每次换用洁净的纸巾,避免交叉污染。 (10)用1ml的洗脱缓冲液洗脱结合的噬菌体。洗脱液可以是含有配体(0.1-1mM)的TBS,或是含有靶蛋白(100μg/ml)的TBS以和固化在平皿上的靶蛋白竞争结合噬菌体。室温下轻缓摇动10-60min。将洗脱液转入微量离心管中。 (10a)或使用通用缓冲液,0.2M甘氨酸-盐酸(PH2.2),1mg/ml BSA。轻缓摇动不超过10min,将洗脱液转入微量离心管中,以150μl 1M Tris-HCl(PH 9.1)中和。 (11)取小量洗脱液(1μl)测定滴度,如果有必要,可将第一轮或第二轮测定滴度的噬斑进行测序(见后续操作)。(未用的洗脱物可4℃保存过夜,第二天进行扩增。在这种情况下,用LB过夜培养ER2537,第二天,用LB培养基以1:100将该培养物稀释至20ml,加入未扩增的洗脱液,在250ml的锥形瓶内,37℃剧烈振摇孵育4.5h,进入步骤13)。 (12)将剩余的洗脱物进行扩增:将洗脱物加入20ml ER2537培养物中(对数早期),37℃剧烈振摇孵育4.5h。 (13)将培养物转入微量离心管中,4℃,10000转离心10min,将上清转入新的离心管中,再次离心。 (14)将80%的上清转入新的离心管中,加入1/6体积的PEG/ NaCl。4℃沉淀噬菌体至

噬菌体展示肽库说明书

ProteIn tooLS Ph.D.? Phage Display Libraries Instruction Manual neB #e8100S, #e8101S, #e8102L, #e8110S, #e8111L, #e8120S, #e8121L Store at –20°C

table of Contents: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Media and Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 General M13 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Construction of pIII-Display Libraries using M13KE . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 Panning Protocols Protocol 1: Surface Panning Procedure (Direct Target Coating) . . . . . . . . .14 Protocol 2: Solution-phase Panning with a Biotinylated Target and Streptavidin Plate Capture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 Protocol 3: Solution-phase Panning with Affinity Bead Capture . . . . . . . . .19 Post Panning Protocols Plaque Amplification for ELISA or Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Sequencing of Phage DNA Rapid Purification of Sequencing Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 Phage ELISA Binding Assay with Direct Target Coating . . . . . . . . . . . . . . . . . . .25 Use of Synthetic Peptides in Specificity Analysis . . . . . . . . . . . . . . . . . . . . . . . . . .27 Expression of Selected Sequences as Monovalent MBP Fusions . . . . . . . . . . . . . . . .27 Appendix: Optimizing Peptide Binding Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29 Choice of Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30 Selection of Cell-Specific Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38 Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 1

噬菌体展示肽库说明说 中文版

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第13卷第1期 2005年1月 中国兽医寄生虫病 Chinese Journal of Veterinary Parasit ol ogy Vol .13No .1 Jan .2005  收稿日期:2004208209 噬菌体展示随机肽库及其在抗原表位研究上的应用 王欣之 傅志强 (中国农科院上海家畜寄生虫病研究所,上海200232) 摘 要:本文主要介绍了噬菌体展示系统的原理,并就噬菌体表面展示系统的不同表达载体类别,分别做了描述,同时讨论该展示系统在抗原表位研究上的应用。关键词:噬菌体展示;随机肽库;抗原表位 中图分类号:Q939.48 文献标识码:A 文章编号:100520868(2005)0120042204 PHAGE D I SPLAY RAN DOM L I BRAR Y AND I TS APPLY I N EP I TO PE RESEARCH WANG W in 2zhi,F U Zhi 2qiang (Shanghai Institute of D o m estic A ni m al Parastitology,Shanghai 200232,China ) Abstract: This paper maj ors in intr oducing the p rinci p le of phage dis p lay syste m ,and describing the s orts of this syste m individually as different exp ress vect or .Furher more,we discusse the app licati on of this syste m es pecial 2ly in ep it ope research . Key words: phage dis p lay;random library;ep it ope 抗原表位(ep it ope )或抗原决定簇(antigen de 2ter m inant )一般有两种类型:一种是连续抗原表位,其氨基酸序列在一级结构上是连续的;另一种是不连续的,由一级结构上不连续的氨基酸残基经过肽链的空间折叠而聚集在一起形成的。研究抗原表位的方法很多,对于连续的抗原表位一般采用合成一系列部分重叠的小肽来确定表位的位置以及每个氨 基酸残基的作用[1] ,对于不连续的抗原表位,则可以采用表面模拟合成(surface si m ulati on synthesis )技术等。这些方法都有很大的局限性:需要预先知道该表位的一级结构的序列或结构信息。 随机肽库(Random pep tides library,RP L )是包括了20种氨基酸所有可能排列的多肽的组合体。按表达载体的不同,随机肽库又分噬菌体、质粒、细菌、核糖体等表面展示随机肽库和化学合成的随机肽库等,其中表面展示的随机肽库及其筛选技术最成熟,应用也最广泛。 1 噬菌体展示的基本原理 S m ith (1985)最先将外源基因插入丝状噬菌体f1的基因Ⅲ,使目的基因编码的多肽能以融合蛋白的形式展示在噬菌体表面,从而创建了噬菌体展示 技术[2] 。 噬菌体展示技术是一种基因表达产物与亲和选择相结合的技术。这项技术的主要原理是将蛋白质或多肽的DNA 序列插入到噬菌体颗粒外壳蛋白的一个结构基因的适当位置,外源基因随外壳蛋白的表达而表达,由于被修饰了的外壳蛋白仍然保持与噬菌体颗粒的兼容性,外源基因产物随病毒颗粒的 重新组装而展示到病毒外壳蛋白的表面[3] 。被展示的多肽或蛋白质可保持相对独立空间结构和生物活性。通过反复的亲和选择和扩增,可直接测定所展示的多肽或蛋白质的某些地区生物活性并分离出带有目的基因的噬菌体。 2 噬菌体表面展示系统 按表达外源蛋白的噬菌体类别的不同,可将噬 菌体展示系统分为丝状噬菌体展示系统、 λ噬菌体

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