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I m m u n i z a t i o n a n d g e n e r a t i o n o f a N a n o b o d y p h a g e d i s p l a y l i b r a r y
1 0 0 mg o f e i t h e r r e c o m b i n a n t m o u s e o r h u m a n V C A M 1 / F c c h i m e r a p r o t e i n ( R n D S y s t e m s , M i n n e a p o l i s , M N ; r e s p . # 6 4 3 - V M a n d 8 6 2 - V C ) w a s r e s u s p e n d e d i n 5 0 0 ml P h o s p h a t e - b u f f e r e d S a l i n e ( P B S ) , m i x e d w i t h 5 0 0 ml G e r b u a d j u v a n t ( L Q # 3 0 0 0 , G E R B U B i o t e c h n i k G m b H , H e i d e l b e r g , G e r m a n y ) a n d i n j e c t e d s . c . i n a c a m e l ( C a m e l u s d r o m e d a r i u s ) t h a t w a s h o u s e d i n t h e C e n t r a l V e t e r i n a r y R e s e a r c h L a b o r a t o r i e s ( C V R L , D u b a i , U n i t e d Arab Emirates). Mouse VCAM1 protein was injected on days 0, 14 and 28 and human VCAM1 protein on days 7, 21 and 35. On day 39, 30 mL anti-coagulated blood was drawn from the jugular vein and diluted with an equal volume of 0.9% (w/v) NaCl. Peripheral Blood Lymphocytes were isolated using Leucosep tubes (Greiner Bio-One, Monroe, NC) and total RNA was extracted using Trizol reagent (Invitrogen, San Diego, CA), both according to manufacturer's instructions.
20 mg o f t o t a l R N A w a s m i x e d w i t h 2 . 5 g o f o l i g o - d T 1 2 - 1 8 a n d c o n v e r t e d i n t o c D N A u s i n g t h e S u p e r s c r i p t I I R e v e r s e T r a n s c r i p t a s e s y s t e m ( I n v i t r o g e n , S a n D i e g o , C A ) . T o t a l c D N A i s u s e d a s t e m p l a t e f o r a p r i m a r y P C R r e a c t i o n w i t h p r i m e r s C A L L 0 0 1 ( 5 - G T C C T G G C T G C TCTTCTACAAGG-3) and CALL002 (5-GGTACGTGCTGTTGAACTGTTCC-3) using the FastStar Taq DNA polymerase system (Roche, Basel, Switzerland). The CALL001 primer anneals to the template strand of leader-signal sequences of both camelid VH and VHH genes, whereas the CALL002 primer anneals to the coding strand of CH2-sequence of camelid IgG. The 700-750 bp DNA fragment (amplicon from heavy-chain-only antibodies) is extracted from a 1% (w/v) agarose gel using the Gel Extraction Kit (Qiagen, Hilden, Germany), avoiding the 1000 bp DNA fragment (amplicon from conventional antibodies), and is used as template for a nested PCR reaction with primer A6E (5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3', R is A or G, PstI restriction enzyme site is underlined) and primer 38 (5'- GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3', NotI restriction enzyme site is underlined). The A6E primer anneals at the template strand for the first 10 codons and primer 38 at the template strand for the last 7 codons of VHH or Nanobody. The resulting circa 400 bp DNA fragment is digested with restriction enzymes PstI and NotI (Roche, Basel, Switzerland), and ligated into the pHEN4 vector ADDIN EN.CITE Arbabi Ghahroudi199723302330233017Arbabi Ghahroudi, M.Desmyter, A.Wyns, L.Hamers, R.Muyldermans, S.Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Sint Genesius Rode, Belgium.Selection and identification of single domain antibody fragments from camel heavy-chain antibodiesFEBS LettFEBS Lett521-64143Amino Acid SequenceAnimalsAntibodies/*geneticsAntibody AffinityAntibody SpecificityBacteriophages/geneticsBinding Sites, AntibodyCamels/*immunologyCloning, MolecularEpitope MappingGene LibraryImmunoglobulin Heavy Chains/*genetics/*immunology/metabolismMolecular Sequence DataPolymerase Chain ReactionRecombinant Proteins/genetics/immunology/metabolism1997Sep 159323027http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9323027first description of pHEN4 plasmid1 using T4 DNA ligase (Fermentas, Burlington, Canada). The pHEN4 vector is a pUC-derived phagemid with an F1 origin of replication and an ampicilin-resistance gene, where Nanobody expression is driven from a Plac promoter , i n d u c e d b y I s o p r o p y l - - D - t h i o g a l a c t o p y r a n o s i d e ( I P T G ) . T h e N a n o b o d y s e q u e n c e s a r e l i g a t e d i n f r a m e a n d d o w n s t r e a m o f a p e l B l e a d e r s i g n a l s e q u e n c e , a n d u p s t r e a m o f a h e m a g g l u t i n i n ( H A ) t a g a n d t h e g e n e I I I f r o m M 1 3 b a c t e r i o p h a g e . T h e p e l B l e a d e r s e q u e n c e ensures directing the Nanobody-fusion protein to the periplasmic region of E. coli.
The whole pHEN4-Nanobody ligation product is electroporated into electrocompetent E. coli TG1 cells using the Gene Pulser electroporation instrument (BioRad, Richmond, CA) and 0.1 cm electroporation cuvettes, plated out on Luria-Broth (LB) solid agar supplemented with 0.1% (w/v) glucose and 100 mg / m L a m p i c i l l i n ( L B / A M P / G L U a g a r ) a n d g r o w n o v e r n i g h t a t 3 7 C . B a c t e r i a l c o l o n i e s w e r e c o l l e c t e d b y s c r a p i n g i n l i q u i d L B m e d i u m t o o b t a i n t h e a n t i - V C A M 1 N a n o b o d y i m m u n e l i b r a r y o f a b o u t 1 0 8 t r a n s f o r m a n t s .
A g e n e r a l s t e p - b y - s t e p p r o t o c o l t o g e n e r a t e a N a n obody immune library is also described elsewhere ADDIN EN.CITE Hassanzadeh Gh20102585258525855Hassanzadeh Gh, G.Saerens, D.Muyldermans, S.Kontermann, R. E.Dubel, S.Isolation of antigen-specific NanobodiesAntibody Engineering251-26622010Berlin-HeidelbergSpringer-Verlag2.
Phage display and biopaning of the anti-VCAM1 Nanobody phage display library.
21010 cells from the anti-VCAM1 Nanobody immune li b r a r y w e r e i n o c u l a t e d i n 3 0 0 m L 2 x T Y m e d i u m s u p p l e m e n t e d w i t h 0 . 1 % ( w / v ) g l u c o s e a n d 1 0 0 mg / m L a m p i c i l l i n ( 2 x T Y / A M P / G L U ) a n d g r o w n a t 3 7 C u n t i l t h e e x p o n e n t i a l p h a s e . 5 1 0 1 1 M 1 3 K O 7 h e l p e r p h a g e s w e r e a d d e d t o i n f e c t b a c t e r i a l c e l l s . A f t e r o v e r n i g h t g r o w t h i n 2 x T Y m e d i u m s u p p l e m e n t e d w i t h 1 0 0 mg / m L a m p i c i l l i n a n d 7 0 mg / m L k a n a m y c i n ( 2 x T Y / A M P / K A N ) , t h e l i b r a r y o f p h a g e - d i s p l a y e d N a n o b o d i e s i s g e n e r a t e d . V i r i o n s a r e h a r v e s t e d f r o m t h e b a c t e r i a l c u l t u r e m e d i u m v i a p r e c i p i t a t i o n : b a c t e r i a l c u l t u r e s w e r e c e n t r i fuged for 30 min at 4C at 2200g, supernatant was collected and mixed with a final concentration of ice-cold 3% (w/v) polyethylene glycol 6000 and 400 mM NaCl (PEG/NaCl). After 30 min incubation on ice, precipitates were centrifuged at 4C for 30 min at 2200g, supernatant was removed completely, and virions were resuspended in 1 mL PBS. OD260nm of a 50-fold dilution of the virion solution was measured and the phage titer was calculated using the following formula:1 OD260nm = 1.51013 phage particles per mL. The phage particle concentration was adjusted to 21012/mL in PBS and used for subsequent biopanning.
As a first step in the biopanning protocol, 100 L of either mouse or human recombinant VCAM1/Fc chimeric protein at 100 g/mL in 100 mM NaHCO3 was immobilized overnight at 4C into one well of a Maxisorb 96-well microtiter plate (NUNC, Rochester, NY) and overcoated with 2% (w/v) skimmed milk powder in PBS for 1h at room temperature. 1011 Nanobody-displaying virions were added for 1h at room temperature a n d u n b o u n d p h a g e s w e r e w a s h e d a w a y w i t h P B S + 0 . 0 5 % ( v / v ) T w e e n - 2 0 ( P B S / T w e e n ) . B o u n d p h a g e s w e r e e l u t e d u s i n g 1 0 0 ml 1 0 0 m M T r i e t h y l a m i n e ( S i g m a - A l d r i c h , S t . L o u i s , M O ) a n d n e u t r a l i z e d i n 1 0 0 L 1 M T r i s - H C l , p H 7 . 4 . H a r v e s t e d p h a g e s w e r e r e - a m p l i f i e d b y infection of exponentially growing E. coli TG1 cells overnight at 37C in the presence of 109 M13KO7 helper phages in 2xTY/AMP/KAN medium. Amplified virions were collected by precipitation, as described before. The biopanning procedure is then repeated for multiple rounds of selection on either mouse or human recombinant VCAM1/Fc chimeric protein. Three types of biopannings were performed: (i) multiple rounds of selections on mouse VCAM1 recombinant protein only; (ii) multiple rounds of selections on human VCAM1 recombinant protein only; (iii) multiple rounds of selections, alternatingly on mouse or human VCAM1 recombinant protein.
A general step-by-step protocol to biopan a phage-displayed Nanobody library is also avaliable elsewhere ADDIN EN.CITE Hassanzadeh Gh20102585258525855Hassanzadeh Gh, G.Saerens, D.Muyldermans, S.Kontermann, R. E.Dubel, S.Isolation of antigen-specific NanobodiesAntibody Engineering251-26622010Berlin-HeidelbergSpringer-Verlag <