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Libraries
General Introduction to
Over the past 10 years Greg Winter’s lab at the MRC Laboratory of Molecular Biology and the MRC Centre for Protein Engineering (Cambridge, UK) has created a number of artificial libraries of antibodies that can be ud to derive binders to almost any target molecule using phage display and lection. The binders can be ud for all the same applications as conventional monoclonal antibodies (ELISA, Western blotting, FACS, immunohistochemistry etc) but can be isolated in a fraction of the time and without the need for animal immunisation. To date the so called “naïve” or “single pot” phage-antibody libraries have been ud successfully in hundreds of molecular biology labs world-wide to derive highly specific antibody reagents to a wide range of different proteins, peptides or small molecule compounds.
The latest libraries (Tomlinson I and J) that are being distributed by the MRC HGMP Resource Centre each compri over 100 million different scFv fragments cloned in an ampicillin resistant phagemid vector and transformed into TG1 E. Coli cells (scFv fragments compri a single polypeptide with the VH and VL domains attached to one another by a flexible Glycine-Serine linker). By carefully following the protocol provided, large numbers of phagemids can be produced and ud to lect specific binders to target molecules that are attached to the surface of a tube or biotinylated and captured by streptavidin coated beads (so called “panning”). After each round of panning, the non-binders are washed away and the phagemids bound to the target molecule/s are eluted and amplified by infection into fresh TG1 cells. After producing new phagemids from the previous round of panning, the process can be repeated. Typically two or three rounds of panning are required to ensure that more than half the different scFvs in the lected population bind to the target molecule. The monoclonal scFvs can then be screened for binding (using a simple ELISA bad protocol) and then ud for further analysis of the target molecule. Since all the functional scFvs in the Tomlinson I and J libraries bind Proteins A and L, either of the condary reagents can be ud for detection, purification or immobilisation. Alternatively, condary reagents that bind the attached myc or HIS6 tags can be ud, although in our experience it is better to u the Protein A or L reagents.
Finally, we would like to emphasi that the libraries reprent a valuable resource. Whether you are familiar with phage display or not we recommend that you perform test lections and subquent ELISA screening using the anti-bovine rum albumin and anti-bovine ubiquitin controls provided. Only when the experiments have been successfully carried out should you defrost the libraries and start preparing library phage.
Both libraries are bad on a single human framework for V H (V3-23/DP-47 and J H4b) and Vκ好听个性签名
(O12/O2/DPK9 and Jκ1) with side chain diversity incorporated at positions in the antigen binding site that make contacts to antigen in known structures and are highly diver in the mature repertoire. The canonical structure (V H: 1-3, Vκ: 2-1-1) encoded by this framework is by far the most common in the human antibody repertoire. The CDR3 of the heavy chain was designed to be as short as possible yet still able to form an antigen binding surface. Both libraries can be lected and affinity matured without knowing the quences of lected clones. Libraries are in phagemid/scFv format and have been pre-screened for binding to Protein A and Protein L so that the majority of clones in the unlected libraries are functional.
Constructed in pIT2 (HIS myc tag). Diversified (DVT) side chains bad mainly on tho positions which are diver in the primary repertoire (total of 18 residues - H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96). After lection, can be matured by incorporating additional diversity bad on somatic mutation.
Library size (with inrt): 1.47 x 108陈小春老婆
Percentage inrt: 96%
As above but with NNK side chains.
Library size (with inrt): 1.37 x 108
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Percentage inrt: 88%
1. Check that you have received:
a tube of Library I (~500 µl)
a tube of Library J (~500 µl)
助力泵a glycerol stock of a positive control anti-ubiquitin ScFv in bacterial strain TG1 (labeled TG1-anti
ubi)
a glycerol stock of a positive control anti-BSA ScFv in bacterial strain TG1 (labeled 13CG2)
a glycerol stock of T-phage resistant E. Coli. TG1 for propagation of phage (labeled TG1Tr)
∆(lac-proAB) supE thi hsdD5/F' traD36 proA+B lacI q lacZ∆M15)
清肠道排宿便最有效的方法(K12
a glycerol stock of E. Coli. HB2151 for expression of antibody fragments
ara ∆(lac-proAB) thi/F' proA+B lacI q lacZ∆M15)
(K12
KM133 (~100 µl with 107 pfu/ml)
Phage
2. Check the library is still frozen and make sure you keep it frozen at -70°C until needed.
3. Make stock of KM13 according to Protocol G.
4. Run through the protocols using the control clones before you u the library: Streak the controls
on TYE plates containing 100 µg/ml ampicillin and 1 % gluco. After overnight growth at 37°C in an incubator pick a single colony from each and grow the overnight (shaking at 37°C) in 5 ml 2xTY1 containing 100 µg/ml ampicillin and 1 % gluco. Make phage for the positive and negative controls parately (u 500 µl of overnight in D1-D10). U a 1:100 mixture of phage produced from the positive and the negative controls and perform one round of lection (C1-C11) using 100 µg/ml of ubiquitin2 in PBS for coating. Check for enrichment of ubiquitin binders (should be over 50% after one round of lection) by monoclonal phage ELISA (E9-E14).
5. Wherever possible u devoted pipettes and disposable plastic ware. The u of polypropylene
tubes is recommended as phage may adsorb non-specifically to other plastics.
6. For efficient infection of phage, E. coli must be grown at 37°C and be in log pha (OD at 600 nm
of 0.4). To prepare this:
i. Transfer a bacterial colony from a minimal media plate into 5 ml of 2xTY medium (no antibiotics
or gluco). Grow shaking overnight at 37°C.
ii. Next day dilute overnight 1:100 into fresh 2xTY medium. Grow shaking at 37°C until OD 600 is
0.4 (1.5-2 hrs)
7. All centrifugations, except tho performed in a micro centrifuge, are performed at 4°C.
8.Libraries I and J must be ud parately and preferably in parallel. This will ensure
lecting the most antigen binding clones.
In advance
Gather all equipment and reagents (product details are given in the notes at the end of all the protocols). Make sure you have all the necessary media and plates for bacterial growth (the large Bio-Assay plates need to be air-dried in a sterile environment for 2 hrs before u).
Plan your time - most of the daily procedures can be performed simultaneously, so read through each protocol carefully before starting.
Steps
Procedure
Day 1 (5 hrs) A1-A6 Grow libraries I and J and make phage
B1-B3 Make condary stock of libraries
Day 2 (6 hrs) A7-A12 Grow libraries I and J and make phage (cont.)
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C1 Coat immunotubes for 1st round of lection
Day 3 (6.5 hrs) C2-C11 1st round of lection
Day 4 (3 hrs) D1-D6 Make phage from 1st round of lection
C1 Coat immunotubes for 2nd round of lection
Day 5 (6.5 hrs) D7-D11 Make phage from 1st round of lection (cont.)
C2-C11 2nd round of lection
Day 6 (3 hrs) D1-D6 Make phage from 2nd round of lection
C1 Coat immunotubes for 3nd round of lection
Day 7 (6.5 hrs) D7-D11 Make phage from 2nd round of lection (cont.)
C2-C11 3rd round of lection
Day 8 (3 hrs) D1-D6 Make phage from 3rd round of lection
E1 Coat 96 well plate for polyclonal phage ELISA
Day 9 (6.5 hrs) D7-D11 Make phage from 3rd round of lection (cont.)
E2-E8 Polyclonal phage ELISA
Further characterisation of individual clones can be performed by monoclonal phage ELISA (protocol E), monoclonal ELISA using soluble scFv fragments (protocol F), PCR screening (to check for inrt, protocol H) and quencing (protocol I).
1. Add the library stock to 200 ml pre-warmed 2xTY containing 100 µg/ml ampicillin and 1 %
gluco.
2. Grow shaking at 37°C until the OD 600 is 0.4 (1-2 hrs).
3. Take 50 ml of this and add 2x1011 KM13 helper phage3. (U the remaining 150 ml to make a
condary bacterial stock of the library by following protocol B).
4. Incubate without shaking in a 37°C water bath for 30 min.
5. Spin at 3,000 g for 10 min (3,600 rpm in Centra 8 or equivalent). Resuspend in 100 ml of 2xTY
containing 100 µg/ml ampicillin, 50 µg/ml kanamycin and 0.1% gluco.
6. Incubate shaking at 30°C overnight.
要幸福就要奋斗7. Spin the overnight culture at 3,300 g for 30 min (4,000 rpm in Centra 8 or equivalent).
8. Add 20 ml PEG/NaCl (20 % Polyethylene glycol 6000, 2.5 M NaCl)to 80 ml supernatant. Mix well
and leave for 1 hr on ice.
9. Spin 3,300 g for 30 min (4,000 rpm in Centra 8 or equivalent). Pour away PEG/NaCl. Respin
briefly and aspirate any remaining dregs of PEG/NaCl.
10 Resuspend the pellet in 4 ml PBS and spin at 11,600 g for 10 min in a micro centrifuge to remove
any remaining bacterial debris.
11. Store the phage at 4°C for short term storage or in PBS with 15 % glycerol for longer term
storage at -70°C.
12. To titre the phage stock dilute 1µl phage in 100µl PBS, 1µl of this in 100µl PBS and so on until
there are 6 dilutions in total. Add 900µl of TG1 at an OD 600 of 0.4 to each tube and incubate at 37°C in a waterbath for 30 mins. Spot 10 µl of each dilution on a TYE5 plate containing 100 µg/ml ampicillin and 1 % gluco and grow overnight at 37°C. Phage stock should be 1012-1013/ml, enough for at least 10 lections.
