Download Supporting Information Large-scale phage display libraries of single

Supporting Information
Large-scale phage display libraries of single-chain camelid antibodies
directed against cell surface antigens of Chlamydomonas reinhardtii
Wenzhi Jiang, Julian N. Rosenberg, Akelia D. Wauchope, Jacqueline M.
Tremblay, Charles B. Shoemaker, Donald P. Weeks, George A. Oyler
Supporting Information Methods
Methods S1 Expression and purification of VHH fusion proteins
Methods S2 VHH protein blot analysis
Supporting Information Figures
Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7
DNA Sequence 1 and DNA Sequence 2
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Supporting Information Figures:
Figure S1. Pre-immune and post-immune responses of an alpaca
immunized with total cell lysates from C. reinhardtii. Protein blot analysis of
total cell protein extracts from Nannochloropsis oceanica (Lane 1), Thalassiosira
pseudonana (Lane 2), Chlamydomonas reinhardtii (Lane 3), Coccomyxa
subellipsoidea (Lane 4), and Chlorella variabilis (Lane 5) used to determine the
presence of antibodies against algal proteins in pre-immune (A pre) and postimmune (B post) sera from an alpaca immunized with total cell proteins from C.
reinhardtii. A 1:1,00 dilution of alpaca sera was used along with a 1:10,000
dilution of secondary antibodies against alpaca antibodies.
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Figure S2. Cassettes for over-expression of C. reinhardtii VHH B11gene
constructs in E.coli. A. Cassette for expression of a VHH B11 fusion protein
containing the Trx A protein at the N-terminus, an internal 6XHIS tag, and an ETag at the C-terminus; B. Cassette for expression of a VHH H10 fusion protein
containing the Trx A protein at the N-terminus, an internal 6XHIS tag, and an ETag at the C-terminus; C. Cassette for expression of a fluorescent fusion protein
containing Trx A protein at the N-terminus, an internal 6XHIS tag followed by the
mCherry fluorescent protein, the VHH B11 coding region and an E-Tag at the Cterminus.
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Figure S3. Amino acid sequence alignments of unique Chlamydomonas
surface binding VHHs.
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Figure S4. Purified VHH proteins (red arrow) directed against
Chlamydomonas cell surface proteins overproduced in E. coli and
visualized by coomassie blue staining of the proteins separated by SDSPAGE. Molecular weight of purified Chlamydomonas VHH proteins ranged from
~33 to ~34.5KD.
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Figure S5. Protein blot s of Chlamydomonas reinhardtii (cc124) cell lysate
proteins. Lanes 1 and 2) Protein blots of cell lysates from the C. reinhardtii wallless mutant CC503 (Lane 1) and from wild-type C. reinhardtii CC124 (Lane 2)
probed with C. reinhardtii VHH B11. Lanes 3-8) Wild-type C. reinhardtii CC124
cell lysates probed with C. reinhardtii VHH antibodies H10, C3, B12, B1, F5 and
F1, respectively. Equal quantities (60 ug) of cell lysates from the wall-less
Chlamydomonas mutant, cc503, (lane 1) and wild-type Chlamydomonas,
cc124, (lanes 2-8) were loaded into each well of the SDS-PAGE used for protein
separation. Each VHH antibody contained an ETAG domain. Anti-ETAG antibody
conjugated with HRP was used to detect VHHs bound to antigens contained in
the protein blots.
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Figure S6. ELISA analyses measuring binding by E-TAG VHH B11 to
Chlamydomonas reinhardtii cell lysate proteins (- -) and inhibition of
binding following preincubation with excess VHH B11 (--). Standard ELISA
assays were conducted except that prior to incubation with E-TAG B11, cell
lysate proteins in microtiter dish wells were incubated with an excess of V HH B11.
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Figure S7. ELISA analyses measuring inhibition of binding by E-TAG VHH
H10 to Chlamydomonas reinhardtii cell lysate proteins following
preincubation with excess VHH H10 (−−) and lack of inhibition of E-TAG
VHH B11 binding by preincubation with an excess of VHH H10 (−−).
Standard ELISA assays were conducted except that prior to incubation with ETAG H10 (or E-TAG B11), cell lysate proteins in microtiter dish wells were
incubated with an excess of VHH B11( or VHH H10). ELISA analyses of E-TAG
VHH H10 (−−) and VHH B11 (− −) binding to cell lysate proteins provided noncompetitive binding standards.
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