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).