【KNOWLEADGE】Amplifying a large phage-display library without losing diversity

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关键词：Amplifyingphage-displaylibrarydiversity: D% {" t1 S6 v6 {% M0 `" l
This section describes how to amplify a library with minimal danger of substantially reducing its diversity.Amplification is accomplished by infecting fresh cells with a portion of the library,growing the infected cells in large cultures,and isolating the phage secreted by the infected cells into the medium.They key to maintaining diversity is to ensure that the number of cells infected (before the cells have had a chance to replicate)is much larger than the number of clones in the primary library.8 I& ?. ~0 [! B: F

2 r! ?: q; j- b3 ^9 T1.Inoculate two 1-liter culture flasks containing 100 ml terrific broth with 1 ml of an overnight culture of K91BluKan.Shake vigorously at 37º until the OD600of a 1/10 dilution reaches ~0.2 (late log phase).
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2.Slow the shaking way down for 5 min to allow sheared F pili to regenerate.$ I0 f6 ]1 f+ E% u1 {
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3.Into each flask add ~1012 physical particles (~5 ×1010 TU)of the library to be amplified.The final concentration of physical particles (1010 virions/ml)is roughly comparable to the concentration of viable host cells.Continue slow shaking for 15 min.
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1 m) ?8 a  s4 W- l5 h- f5 i4.Pour each culture into a pre-warmed 3-liter fernbach flask containing 1 liter of NZY supplemented with 0.22 mg/ml tetracycline; shake vigorously for 35 min at 37℃.
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5.To each flask add 1 ml 20 mg/ml tetracycline,bringing the concentration up to 18 mg/ml.If the library is in the f88-4 vector,also add 1 ml 1 M IPTG to induce expression of the recombinant (peptide-bearing)gene VIII.' [0 U6 u* k( v. z, x$ X' J" l

/ Y- \9 E9 E% G! H& }# z$ ^6.Remove a 7-µl sample from each flask for the dilutions (next step),then continue shaking the flasks vigorously overnight.  M! B) P8 H; `/ d1 C

* q! p# p' q  M- g7 e7.Spread 200 µl of 10-4 and 10-5 serial dilutions of the two 7-µl samples from the previous step (diluent = NZY)on NZY plates containing 40 mg/ml tetracycline and 100 mg/ml kanamycin.Count the colonies the next day.A colony count of ~100 on the 10-5 plates indicates ~5 ×1010 infected cells per culture (~5% of the input physical particles--a typical infectivity for fd-tet phage).The 1011 infected cells in both flasks are enough for every clone in a billion clone initial library to be represented by 100 infected cells in the amplification—presumably sufficient over-representation to preserve essentially all the diversity.% I+ p: P) N1 l
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8.Purify virions from the cultures as in steps 2–4,6–7 and 9–20 of VirionPurification.DOC,omitting the detergent treatment.+ M+ q" H/ Z6 {, {
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9.We usually sequence a sample of the phage as described in sequencing.doc to confirm the degenerate region of the coding sequence.

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Phage display4 W5 D& E. u; U% A) m3 N$ e9 Z
From Wikipedia, the free encyclopedia6 K$ u0 V' N3 p0 h4 B4 R
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Phage display is a method for the study of protein-protein, protein-peptide, and protein-DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them.[1] Phage Display was originally invented by George P. Smith in 1985 and he demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene3 of filamentous phage.[1] This technology was further developed and improved by various other people like John McCafferty for display of proteins like antibodies for therapeutic protein engineering. The connection between genotype and phenotype enables large libraries of proteins to be screened and amplified in a process called in vitro selection, which is analogous to natural selection. The most common bacteriophages used in phage display are M13 and fd filamentous phage,[2][3] though T4[4], T7, and λ phage have also been used.
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Contents [hide]* w5 V( j% O3 i/ L( ~; m) t
1 Principle . z" v5 j' s: U) C
2 General protocol & F' |; b  o3 J! e5 B
3 Applications 7 ~: G% S' {' F( O) m
4 See also " T4 K+ D) u7 L9 n: b7 E% M  L
5 References
: g) K7 k+ i+ r$ {" D! L6 See also
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[edit] Principle
* q& R- h# J4 v, GLike the two-hybrid system, phage display is used for the high-throughput screening of protein interactions. In the case of M13 filamentous phage display, the DNA encoding the protein or peptide of interest is ligated into the pIII or pVIII gene. Multiple cloning sites are sometimes used to ensure that the fragments are inserted in all three possible frames so that the cDNA fragment is translated in the proper frame. The phage gene and insert DNA hybrid is then transformed into E. coli bacterial cells such as TG1, SS320, ER2738, or XL1-Blue E. coli. If a "phagemid" vector is used (a simplified display construct vector) phage particles will not be released from the E. coli cells until they are infected with helper phage, which enables packaging of the phage DNA and assembly of the mature virions with the relevant protein fragment as part of their outer coat on either the minor (pIII) or major (pVIII) coat protein. The incorporation of many different DNA fragments into the pIII or pVIII genes generates a library from which members of interest can be isolated." A. K& b! o1 k* c

8 @3 e  [. B4 oBy immobilizing a relevant DNA or protein target(s) to the surface of a well, a phage that displays a protein that binds to one of those targets on its surface will remain while others are removed by washing. Those that remain can be eluted, used to produce more phage (by bacterial infection with helper phage) and so produce a phage mixture that is enriched with relevant (i.e. binding) phage. The repeated cycling of these steps is referred to as 'panning', in reference to the enrichment of a sample of gold by removing undesirable materials.
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Phage eluted in the final step can be used to infect a suitable bacterial host, from which the phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the relevant, interacting proteins or protein fragments.- j& e2 _& p4 o4 Y3 B

* S# ^5 d7 |3 K/ r' LRecent work published by Chasteen et al., shows that use of the helper phage can be eliminated by using a novel 'bacterial packaging cell line' technology.[5]
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) S1 y- o, O' N$ A: d8 f7 I( u$ }% k[edit] General protocol
6 y7 |3 ~4 s. y0 i' }Target proteins or DNA sequences are immobilised to the wells of a microtiter plate.   m- x9 u- D7 q5 R8 u- i
Many genetic sequences are expressed in a bacteriophage library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage. " g5 p" S% s3 K
This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
7 }) \% C- t7 @. h0 n/ APhage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
# P+ P! \2 ^! l5 a2 J$ X8 T& |Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant phage (i.e. non-binding) than were present in the initial mixture. : z, [" v& |8 T6 ~! u' J9 u
The DNA within the interacting phage contains the sequences of interacting proteins, and following further bacterial-based amplification, can be sequenced to identify the relevant, interacting proteins or protein fragments.
& A) X' e* Q: d6 e) C[edit] Applications4 W  u5 h2 j7 o: V' P2 `; Z
The applications of this technology include determination of interaction partners of a protein (which would be used as the immobilised phage "bait" with a DNA library consisting of all coding sequences of a cell, tissue or organism) so that new functions or mechanisms of function of that protein may be inferred[6]. The technique is also used to determine tumour antigens (for use in diagnosis and therapeutic targeting)[7] and in searching for protein-DNA interactions[8] using specially-constructed DNA libraries with randomised segments.
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Phage display is also a widely used method for in vitro protein evolution (also called protein engineering). As such, phage display is a useful tool in drug discovery. It is used for finding new ligands (enzyme inhibitors, receptor agonists and antagonists) to target proteins.[9][10][11].& R/ B" l- ?6 D# ]
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Invention of antibody phage display by John McCafferty in 1990 revolutionised the drug discovery by allowing the display of antibody fragments on phage. [12] Antibody libraries displaying millions of different antibodies on phage are frequently used in the pharmaceutical industry for isolation of highly specific therapeutic antibody leads, for development into primarily anti-cancer or anti-inflammatory antibody drugs. One of the most successful was HUMIRA (adalimumab), discovered by Cambridge Antibody Technology as D2E7 and developed and marketed by Abbott Laboratories. HUMIRA, an antibody to TNF alpha, was the world's first fully human antibody[13], which achieved annual sales exceeding $1bn [14] therefore achieving blockbuster status[15].  Q& F6 |" r" I2 T" M2 `9 P  L) m
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Competing methods for in vitro protein evolution are yeast display, bacterial display, ribosome display, and mRNA display.