25. Kurland CG. Dong H: Bacterial growth inhibition by overproduction of 2
protein. Mol Microbiol 1996, 21: 1-4.
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26. Rosenberg AH, Goldman E, Dunn JJ, Studier FW, Zubay G: Effects of 4
consecutive AGG codons on translation in Escherichia coli, demonstrated 5
with a versatile codon test system. J Bacteriol 1993, 175: 716-22.
6
27. Rosano GL, Ceccarelli EA: Rare codon content affects the solubility of 7
recombinant proteins in a codon bias-adjusted Escherichia coli strain.
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Microb cell fact 2009, 8: 41-49.
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28. Gvritishvili AG, Leung KW, Tombran-Tink J: Codon preference 10
optimization increases heterologous PEDF expression. PLoS One 2010, 5:
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e15056.
12 13 14 15
Figure Legends 1
Figure 1. Schematic diagram of the constructs used for TAT-Apoptin protein 2
expression. (A) Schematic representation of the TAT-Apoptin protein fused with 3
different affinity tags together with the expression vectors used in this study. The 4
designations of the TAT-Apoptin protein and its expression vectors are indicated, 5
(a), (b), (c) and (d). The constructs, (a) and (b), contain the full-length TAT-VP3 6
gene cloned into the vectors pET28a and pGEX-4T-1; these were used for 7
expression of TAT-Apoptin protein with either a six-histidine (6×His) tag or a 8
glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs (c) 9
and (d) containing the TAT-VP3 gene that was codon-optimized; this was derived 10
from construct (b) by replacing rare codons without altering the amino acid 11
sequence. The codon-optimized TAT-VP3gene, TAT-VP3opt, was then cloned into 12
pET28a and pGEX-4T-1. (B) Sequence comparison between the TAT-VP3 gene 13
and the TAT-VP3opt gene. The nucleotide sequences were compared between the 14
original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized 15
TAT-VP3 gene (TAT-VP3Opt) over the whole coding region. An asterisk (*) 16
represents the fact that the aligned nucleotides are identical.
17 18
Figure 2. Expression of recombinant TAT-Apoptin protein in three different E.
19
coli strains. The TAT-Apoptin protein expression in three E. coli strains, 20
BL21(DE3), BL21(DE3)pLysS and BL21(DE3)CodonPlus-RP, which were 21
transformed with either pET-TAT-VP3 or pGEX-TAT-VP3 and cultivated at 37oC.
22
His-TAT-Apoptin and GST-TAT-Apoptin protein were examined and detected 23
using SDS-PAGE (A, C) and Western-blotting (B, D). Anti-His and anti-GST tag 24
monoclonal antibodies was respectively used to recognize the His-TAT-Apoptin 25
protein and GST-TAT-Apoptin. Lane M, pre-stained protein marker; the symbols 1
“－” and “＋” represented pre-induction and post-induction with 1mM of IPTG 2
over 4 hrs of cultivation in E. coli, respectively.
3 4 5
Figure 3. Productivities of TAT-Apoptin protein and the growth curves of the 6
three recombinant E. coli strains. The productivities of His-TAT-Apoptin (A) and 7
GST-TAT-Apoptin (C) for the three E. coli strains, BL21(DE3), BL21(DE3)pLysS, 8
and BL21(DE3)CodonPlus-RP containing pET-TAT-VP3 or pGEX-TAT-VP3 are 9
shown over the time course of cultivation at 37oC after IPTG induction. The 10
growth curves of BL21(DE3), BL21(DE3)CodonPlus-RP and BL21(DE3)pLysS 11
expressing His-TAT-Apoptin (B) and GST-TAT-Apoptin (D), respectively, in LB 12
medium post-induction.
13 14
Figure 4. Expression of recombinant TAT-Apoptinopt protein in the different E.
15
coli strains. The TAT-Apoptinopt protein was expressed in the E. coli strains 16
BL21(DE3) and BL21(DE3)pLysS, which contained either pET-TAT-VP3opt or 17
pGEX-TAT-VP3opt, at 37oC. His-TAT-Apoptinopt and GST-TAT-Apoptinopt protein 18
were examined and detected using SDS-PAGE (A, C) and Western-blotting (B, D).
19
Anti-His and anti-GST tag monoclonal antibodies was respectively used to 20
recognize the His-TAT-Apoptinopt protein and GST-TAT-Apoptinopt. Lane M, 21
pre-stained protein marker; “ － ” and “ ＋ ” represented pre-induction and 22
post-induction with 1mM of IPTG over 4 hrs of cultivation in E. coli, respectively.
23 24 25
Figure 5. Productivities of TAT-Apoptinopt protein and the growth curves of two 1
recombinant E. coli strains. The productivities of His-TAT-Apoptinopt (A) and 2
GST-TAT-Apoptinopt (C) using two E. coli strains, BL21(DE3) and 3
BL21(DE3)pLysS containing either pET-TAT-VP3opt or pGEX-TAT-VP3opt, 4
respectively, are shown over a time course after IPTG induction at 37oC. The 5
growth curves of BL21(DE3) and BL21(DE3)pLysS expressing 6
His-TAT-Apoptinopt (B) and GST-TAT-Apoptinopt (D) in LB medium 7
post-induction.
8 9
Figure 6. Solubility of E. coli-expressed GST-TAT-Apoptinopt protein under using 10
various cultivation parameters during protein induction. The solubility of 11
GST-TAT-Apoptinopt was determined in the BL21(DE3) strain at different 12
cultivation temperatures (A) and in the presence of various concentrations of 13
IPTG (B, C).
14 15
Figure 7. Productivities of TAT-Apoptinopt protein and the growth curves in two 16
recombinant E. coli strains. The productivities of GST-TAT-Apoptinopt using two E.
17
coli strains, BL21(DE3) and BL21(DE3)pLysS containing pGEX-TAT-VP3opt are 18
shown over time after IPTG induction at 25oC (A). Growth curves of BL21(DE3) 19
and BL21(DE3)pLysS expressing GST-TAT-Apoptinopt, respectively, in LB 20
medium post-induction (B).
21 22
Figure 8. Purification of recombinant GST-TAT-Apoptinopt protein. SDS-PAGE(A) 23
and Western-blot (B) analysis of the GST-TAT-Apoptinopt protein contained in 24
various elution fractions collected from the GSTrap FF affinity column. The 25
cytosolic extract of E. coli strain BL21(DE3) expressing GST-TAT-Apoptinopt
1
protein was loaded onto a GSTrap FF column and the bound protein was eluted 2
with elution buffer as described in Material and Methods. The eluted protein from 3
the GSTrap FF affinity column was analyzed by SDS-PAGE and Western blotting 4
using monoclonal anti-GST antibody. (C) Antigenicity analysis of 5
GST-TAT-Apoptinopt. The purified GST-TAT-Apoptinopt was assayed by Western 6
blotting using positive CAV-infected chicken serum. Lane M, pre-stained protein 7
marker; lane 1, flow through; lane 2, fraction obtained after column washing, lane 8
3 and 4, eluted fraction 1 and 2, respectively, collecting after column elution.(D) 9
Identity of the GST-opt-VP1 protein determined by MALDI-TOF. The bold letters 10
represent actual amino acid matches to published amino acid sequence (Accession 11
No. AF212490) . 12
13
Figure 9. Induction of apoptosis in HL-60 cells by GST-TAT-Apoptinopt. An 14
apoptotic assay of HL-60 cells was performed by flow cytometry after 90 ug/ml of 15
the purified E. coli-expressed recombinant GST-TAT-Apoptinopt protein was 16
co-cultured with HL-60 cell for 24 hours (D). Non-apoptosis controls, (A), (B) 17
and (C), were assayed. These represent respectively HL-60 cells only, HL-60 cells 18
co-cultured with protein buffer and HL-60 cells co-cultured with GST protein. The 19
apoptosis induced by CHX was used as a positive apoptosis control.
20 21
Table 1. Summary of the productivities of the various TAT-Apoptin proteins 22
expressed in the range of E. coli strains.
23