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Hyper-Production of Large Proteins of Spider Dragline Silk Masp2 By Process Biochemistry 51 (2016) 484–490 Contents lists available at ScienceDirect Process Biochemistry jo urnal homepage: www.elsevier.com/locate/procbio Hyper-production of large proteins of spider dragline silk MaSp2 by Escherichia coli via synthetic biology approach 1 1 ∗ ∗ Yan-Xiang Yang , Zhi-Gang Qian , Jian-Jiang Zhong , Xiao-Xia Xia State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China a r a t i b s c t l e i n f o r a c t Article history: Spider dragline silk exhibits excellent mechanical properties that make it a promising protein polymer Received 10 December 2015 for industrial and biomedical applications. Since farming spiders is not feasible due to their highly ter- Received in revised form 16 January 2016 ritorial nature, recombinant production of dragline silk proteins in a foreign host has received great Accepted 22 January 2016 attention. However, their production titer remains low, because efficient expression of very large, highly Available online 27 January 2016 repetitive, glycine-rich silk proteins is a challenge. This work demonstrates the design and high-level production of large dragline silk proteins of major ampullate spidroin 2 (MaSp2) in Escherichia coli by Keywords: synthetic biology approach. The expression levels of MaSp2 with molecular weight of 28.3–256.5 kDa Spider dragline silk were significantly elevated by down-shifting the induction temperature. The beneficial effect was found Escherichia coli to be at least partially attributed to the improved plasmid maintenance in the recombinant cells. Combi- Recombinant protein production Repetitive protein nation of induction temperature downshift with the glycyl-tRNA pool increase in E. coli led to enhanced −1 High cell density cultivation biosynthesis of glycine-rich silk proteins. A high production titer of about 3.6 g l of a 201.6-kDa MaSp2 Synthetic biology approach protein was achieved in a 3-L fed-batch bioreactor, which was the highest as reported. The developed approach may be useful to cost-effective large-scale production of silk proteins. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction a promising protein polymer for numerous industrial application, and it also possesses a great potential in medical and pharmaceuti- Spider dragline silk is a promising and outstanding protein cal areas for its biodegradable and biocompatible features [10–16]. polymer, which exhibits extraordinary mechanical properties of Unfortunately, natural dragline silk of spiders could not be readily superior strength and toughness that make it outperform most of obtained by farming spiders because of their highly aggressive and the natural and other synthetic fibers [1,2]. It is composed almost territorial nature [17]. Numerous attempts have thus been made to entirely of two proteins, major ampullate spidroins 1 (MaSp1) produce recombinant spider dragline silk proteins in various organ- and 2 (MaSp2) [3,4]. Both MaSp1 and MaSp2 are with large isms such as Escherichia coli, yeast, tobacco, mammalian cells, silk molecular weights at 250–320 kDa, consisting of a long, repetitive worm, and even transgenic animals [18–23]. core domain flanked by non-repetitive N-terminal and C-terminal So far, there have been some reports on the production of recom- sequence on both sides [5–7]. And they differ in the composition binant spider dragline silk proteins, but their molecular weights of structural motifs that affect the mechanical properties of the silk were generally below 100 kDa, which is about half of what spiders fibers. MaSp1 is composed of a polyA region, which contributes to make. In addition, the production titers of the recombinant silk pro- the tensile strength of silk, and of a GGX (X = L, Y, Q, or A) motif, teins were still low, typically on the order of 10–100 mg/l with one which is believed to affect fiber formation, while MaSp2 consists of exception of 2.7 g/l for one MaSp1 (Table 1). But, most MaSp1 pro- a polyA region similar to MaSp1, and of a GPGXX repetitive region teins were only produced at ∼0.5 g/l by metabolically-engineered (X = G, Q, Y) which is responsible for the silk elasticity by forming a E. coli as reported by Xia et al. [10]. The results underscored a beta-spiral [4,5,8,9]. Due to its outstanding properties, spider silk is limitation of glycyl-tRNA pool within the host upon silk protein expression [10]. An expression level of 256 mg/l was reported for a 56.2-kDa recombinant MaSp2 protein [24]. As the expression of ∗ very large, highly repetitive, glycine-rich silk proteins is indeed Corresponding authors. Fax: +86 21 34204831. E-mail addresses: [email protected] (J.-J. Zhong), [email protected] challenging, “smart” engineering approaches are urgently required (X.-X. Xia). for further improvement on the production titer, especially for 1 Both of these authors contributed equally to this work. http://dx.doi.org/10.1016/j.procbio.2016.01.006 1359-5113/© 2016 Elsevier Ltd. All rights reserved. Y.-X. Yang et al. / Process Biochemistry 51 (2016) 484–490 485 Table 1 To overexpress a recombinant silk protein encoding 96 repeats Typical examples of recombinant MaSp production. of MaSp1 consensus sequence, plasmid pET19b-MaSpI96 was made −1 Protein Size (kDa) Production level (l ) Ref. from plasmid pET19b-MaSpI4, using the iterative polymerization strategy as described earlier [42]. MaSp1 100.7–284.9 0.5–2.7 g [10] MaSp2 28–53 2–10 mg [18] Plasmid pTetgly2 was constructed to overexpress the glyVXY Gly MaSp1 65–163 0.663 g [19] genes encoding tRNA under the control of the constitutive tet MaSp1, MaSp2 65–163 0.3 g [20] promoter as previously described [10]. This plasmid allowed ele- MaSp1, MaSp2 48–75 ∼120 mg [21] Gly vated pools of tRNA within the harboring strains of E. coli [10]. ADF3, MaSp2 59–140 25–50 mg [22] MaSp2 12.9–99.8 0.5% of total protein [23] ADF3 56.2 256 mg [24] 2.2. Expression of recombinant silk proteins in flask cultivation MaSp1, MaSp2 14.7–41.3 2–15.7 mg [25] ADF3, ADF4 11.9–59.3 10 –360 mg [26] MaSp2 201.6 3.6 g This work Competent cells of E. coli BL21(DE3), a common expression host for pET expression system, were transformed with the desired plasmids and plated on selective LB agar plates containing the native-size MaSp2 of large molecular weight, towards a commer- appropriate antibiotics. Antibiotics were added at the follow- −1 −1 ␮ ␮ cially viable silk protein biomanufacturing process [20,27,28]. ing concentrations: 50 g ml of Km, 35 g ml of Cm, and −1 ␮ Induction temperature is generally an important factor for 50 g ml of Ap when necessary. A single colony was inoculated recombinant protein expression, and it has been widely used to into a 15-ml tube containing 2 ml of LB medium and cultured ◦ improve the expression of soluble protein expression but has dif- overnight at 30 C and 220 rpm in a shaking incubator. This seed ␮ ferent effects on the production level of different proteins in E. coli culture (200 l) was transferred into a 250-ml shake flask con- −1 [29–41] (Table 2). In this study, we reported high-level production taining 20 ml of either R/2 medium supplemented with 10 g l of ◦ of large spider dragline silk MaSp2 protein of 201.6 kDa by modu- glucose or LB medium, and grown at 30 C and 220 rpm. The mini- −1 −1 lating the induction temperature. Production level of recombinant mal R/2 medium (pH 6.80) contains 2 g l of (NH4)2HPO4, 6.75 g l −1 −1 · spider silk proteins was significantly improved by downshifting the of KH2PO4, 0.85 g l of citric acid, 0.7 g l of MgSO4 7H2O, and −1 induction temperature. Furthermore, we performed the induction 5 ml l of a trace metal stock solution [43]. Cell growth was temperature-shift strategy in bioreactors for the MaSp2 production monitored by measuring the absorbance at 600 nm (OD600) with by a metabolically-engineered E. coli with both pET28a-MaSpII64 a BioPhotometer plus spectrophotometer (Eppendorf, Hamburg, ∼ and pTetgly2, a tRNAGly overexpression plasmid. Finally, a higher Germany). When cell OD600 reached 0.4, the cultures were ␤ production titer than ever reported was achieved in a 3-L fed-batch exposed to 1 mM isopropyl- -D-thiogalactoside (IPTG; Sigma, St. bioreactor. Louis, MO), and incubated at the desired temperatures ranging from ◦ 16 to 37 C. Samples of the cell cultures were taken immediately prior to the addition of IPTG and at the desired time after induction 2. Materials and methods for plasmid maintenance and SDS-PAGE analysis. For the SDS-PAGE analysis of recombinant silk proteins, cells were collected by cen- 2.1. Construction of recombinant plasmids ◦ ◦ trifugation at 4 C and 13,000 × g for 10 min and stored at −80 C before further analysis. DNA manipulations were performed according to standard molecular biology protocols. E. coli DH5␣ (Invitrogen Corp., Carls- bad, CA) was used for general gene cloning studies. Cells were 2.3. Production of recombinant silk proteins by high density cell ◦ routinely grown at 30 C in Luria-Bertani (LB) medium (per liter: cultivation in bioreactors 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl). Antibi- −1 otics were added at the following concentrations: 50 ␮g ml The high density cell cultivation of recombinant E. coli cells was −1 ␮ of kanamycin (Km), 35 g ml of chloramphenicol (Cm), and performed in a 3-L stirred bioreactor (BIOFLO 110; New Brunswick −1 ␮ 50 g ml of ampicillin (Ap) when necessary.
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