Production of silk-like proteins,
TS[(AGSGAG)3AS]nRKLPDAS and
TS[(AGSGAG)3AS]n byEscherichia coli.
The recombinant silk-like proteins
TS[(AGSGAG)3AS]nRKLPDAS (n = 2 and
4) and TS[(AGSGAG)3AS]n (n = 2 and
4) were prepared according to the method described previously
(Yanagisawa et al., 2007; M. Yang & Asakura, 2005; Mingying Yang et
al., 2009; M. Yang, Muto, et al., 2008; M. Yang, Tanaka, et al., 2008;
M. Yang et al., 2007). Here, (AGSGAG)3 (Bc3) and RKLPDA
represent the crystalline region of SF and minimal sequence of titanium
binding peptide, respectively. The construction scheme is shown in
Figure 1a. The pUC-Bc3 is the recombinant vector, in which a single unit
of DNA sequence coding (AGSGAG)3AS was inserted into
pUC118 vector plasmid (Yanagisawa et al., 2007; M. Yang & Asakura,
2005). Because this unit contains Spe I and Nhe I
restriction enzyme recognition sites, and because these enzymes produce
complemental sticky ends, directional multiplexing of the unit is
possibly though repeated cut-and-paste manipulations (Prince, McGrath,
DiGirolamo, & Kaplan, 1995; M. Yang & Asakura, 2005). In this study,
we constructed 2-mer and 4-mer of
[(AGSGAG)3AS]n repeats, which were
named pUC-(Bc3) 2 and pUC-(Bc3) 4,
respectively. To add RKLPDA motif at the end of repeats of
(AGSGAG)3AS, oligonucleotide KY-1775,
pCTAGTCGGAAGCTGCCCGACG, and pCTAGCGTCGGGCAGCTTCCGA were annealed and
ligated into the Nhe I site of pUC-(Bc3) 2 and
pUC-(Bc3) 4 to obtain pUC-(Bc3) 2T and
pUC-(Bc3) 4T. Integrities of constructed artificial
genes were confirmed by sequencing using the Dye Terminator Cycle
Sequencing Quick Start Kit (Beckman Coulter, Brea, CA). For the
expression in Escherichia coli , the inserts of these four
plasmids were digested by Bam HI and Hind III, and were
re-cloned into the same sites of the expression vector pET30a (Nobagen
Inc Darmstadt, Germany) to obtain pET-(Bc3)2,
pET-(Bc3)4, pET-(Bc3)2T, and
pET-(Bc3)4T. In pET30a vectors, artificial silk fibroin
proteins are expressed as the fusion proteins that carry a vector
derived 6xHis-tag sequence at the N-terminal.
Because the pET30a vector is driven by a T7 promoter, the expression
vectors were introduced in the E. coli strain BL21(DE3)pLysS
(Nobagen Inc.), which has T7 polymerase, and the transformed cells were
grown in 200–500 mL of LB (Sambrook, Fritsch, & Maniatis, 1989)
containing kanamycin and chloramphenicol at 37 °C. At
OD660 = 0.5, isopropyl β-D-1 thiogalactopyranoside
(IPTG) was added to a final concentration of 1 mM to induce protein
expression for 4 h. After induction, cells were harvested and frozen and
stored at −80 °C until purification. The protein expression was induced
at 25 °C by adding IPTG to a final concentration of 1 mM. The
recombinant proteins were purified by nickel-chelate chromatography
using an ÄKTA explorer 10S/100 (GE Healthcare, Waukesha, WI, USA) column
according to the manufacturer’s protocol. The optimized conditions for
elution were 15 mM imidazole, pH 4.5, and 50 °C. The eluted protein
solutions (6–8 mL) were dialyzed against 10 mM
NaH2PO4, pH 8.0 and 100 mM NaCl using a
Slide-A-Lyzer Dialysis Cassette (3,500 MWCO, Pierce, Waltham, MA),
followed by concentration into 500 µL by using Amicon Ultra
Centrifugation Filters-4 (Merck Millipore, Billerica, MA). The purified
proteins from pET-(Bc3)2, pET-(Bc3)4,
pET-(Bc3)2T, and pET-(Bc3)4T were termed
(Bc3) 2, (Bc3) 4, (Bc3)2T, and (Bc3) 4T, respectively (Figure 1
a).