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Supplementary figure S10.
Construction and expression of codon-modified Stx2eB genes. (A) Schematic
representation of Stx2eB gene for transient expression. p35S, Cauliflower mosaic virus
35S RNA promoter; NOS-T, transcription terminator from A. tumefaciens nopaline
synthase gene; NtADH, 5′-untranslated region (UTR) of tobacco (Nicotiana tabacum)
alcohol dehydrogenase gene; SP, signal peptide for ER translocation derived from
tobacco -D-glucan exohydrolase; HA, HA peptide tag to detect the Stx2eB protein
(Tyr-Pro-Tyr-Asp-Tyr-Pro-Asp-Tyr-Ala);
HDEL,
ER
retention
signal
(Ser-Glu-His-Asp-Glu-Leu). Amino acid sequences of mature Stx2eB protein and
corresponding nucleotide sequences of original Stx2eB gene are shown (B). mStx2eB1
(C) was designed not to contain A/U repeat sequences more than four nucleotides in
length because A/U rich sequences are often recognized as mRNA processing signals.
For example, the AUUUA sequence destabilizes RNA, and the AAUAAA sequence can
be a premature polyadenylation signal. The codon usage database
(http://www.kazusa.or.jp/e/resources/database.html) was used to design a modified
mStx2eB2 (D) that contains codons that are frequently used in lettuce genes. Specifically,
mStx2eB4 (E) was designed to contain codons that are frequently used in lettuce
housekeeping genes (actin, β-tubulin 1, 2, and 3 genes). mStx2eB5 (F) was designed to
have the highest possible GC content. The construct encoding Stx2eB with the original
codons is called Stx2eB in this study and is the same as ADH-ER-Stx2eB in our
previous report (Matsui et al., 2009). (G) Transient expression of Stx2eB constructs in
lettuce protoplasts. Protein samples prepared from protoplasts transfected with the
Stx2eB constructs were separated by SDS-PAGE, blotted onto PVDF membranes, and
probed with an anti-HA antibody. Triangles indicate signals corresponding to Stx2eB.
(B)
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GCGGCGGATTGTGCTAAAGGTAAAATTGAGTTTTCCAAGTATAATGAGGATAATACCTTTACTGTGAAGGTGTCAGGAAGAGAATACTGGACGAACAGATGGAAT
A A D C A K G K I E F S K Y N E D N T F T V K V S G R E Y W T N R W N
115
125
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155
165
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185
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205
TTGCAGCCATTGTTACAAAGTGCTCAGCTGACAGGGATGACTGTAACAATCATATCTAATACCTGCAGTTCAGGCTCAGGCTTTGCCCAGGTGAAGTTTAAC
L Q P L L Q S A Q L T G M T V T I I S N T C S S G S G F A Q V K F N
(C)
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GCAGCAGATTGCGCTAAGGGTAAGATTGAGTTCTCCAAGTACAACGAGGATAACACCTTCACAGTGAAGGTGTCAGGAAGGGAGTACTGGACAAACAGGTGGAAC
A A D C A K G K I E F S K Y N E D N T F T V K V S G R E Y W T N R W N
115
125
135
145
155
165
175
185
195
205
TTGCAACCATTGTTGCAAAGCGCTCAACTCACAGGGATGACAGTGACAATCATCTCTAACACCTGCAGCTCAGGGTCAGGGTTCGCCCAAGTGAAGTTCAAC
L Q P L L Q S A Q L T G M T V T I I S N T C S S G S G F A Q V K F N
(D)
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GCAGCAGATTGTGCAAAAGGTAAAATTGAATTTTCTAAATATAATGAAGATAATACATTTACAGTTAAAGTTTCTGGTAGAGAATATTGGACAAATAGATGGAAT
A A D C A K G K I E F S K Y N E D N T F T V K V S G R E Y W T N R W N
115
125
135
145
155
165
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185
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CTTCAACCACTTCTTCAATCTGCACAACTTACAGGTATGACAGTTACAATTATTTCTAATACATGTTCTTCTGGTTCTGGTTTTGCACAAGTTAAATTTAAT
L Q P L L Q S A Q L T G M T V T I I S N T C S S G S G F A Q V K F N
(E)
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GCCGCCGATTGCGCCAAGGGTAAGATCGAATTCTCCAAGTACAACGAAGATAACACTTTCACTGTTAAGGTTTCCGGTCGTGAATACTGGACTAACCGTTGGAAC
A A D C A K G K I E F S K Y N E D N T F T V K V S G R E Y W T N R W N
115
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135
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155
165
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CTCCAACCACTCCTCCAATCCGCCCAACTCACTGGTATGACTGTTACTATCATCTCCAACACTTGCTCCTCCGGTTCCGGTTTCGCCCAAGTTAAGTTCAAC
L Q P L L Q S A Q L T G M T V T I I S N T C S S G S G F A Q V K F N
(F)
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GCGGCGGACTGCGCGAAGGGCAAGATCGAGTTCTCGAAGTACAACGAGGACAACACGTTCACGGTCAAGGTCTCGGGCCGCGAGTACTGGACGAACCGCTGGAAC
A A D C A K G K I E F S K Y N E D N T F T V K V S G R E Y W T N R W N
115
125
135
145
155
165
175
185
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205
CTGCAGCCGCTGCTGCAGTCGGCGCAGCTGACGGGCATGACGGTCACGATCATCTCGAACACGTGCTCGTCGGGCTCGGGCTTCGCGCAGGTCAAGTTCAAC
L Q P L L Q S A Q L T G M T V T I I S N T C S S G S G F A Q V K F N
(G)
1
2
4
5
2eB tx2eB tx2eBtx2eB 2eB
x
t
mS mS
mS mS
Stx
45
30
20.1
14.3
6.5
kDa