Secretory production of tetrameric native full-length streptavidin with thermostability using Streptomyces lividans as a host

Background Streptavidin is a tetrameric protein derived from Streptomyces avidinii, and has tight and specific biotin binding affinity. Applications of the streptavidin-biotin system have been widely studied. Streptavidin is generally produced using protein expression in Escherichia coli. In the present study, the secretory production of streptavidin was carried out using Streptomyces lividans as a host. Results In this study, we used the gene encoding native full-length streptavidin, whereas the core region is generally used for streptavidin production in E. coli. Tetrameric streptavidin composed of native full-length streptavidin monomers was successfully secreted in the culture supernatant of S. lividans transformants, and had specific biotin binding affinity as strong as streptavidin produced by E. coli. The amount of Sav using S. lividans was about 9 times higher than using E. coli. Surprisingly, streptavidin produced by S. lividans exhibited affinity to biotin after boiling, despite the fact that tetrameric streptavidin is known to lose its biotin binding ability after brief boiling. Conclusion We successfully produced a large amount of tetrameric streptavidin as a secretory-form protein with unique thermotolerance. Electronic supplementary material The online version of this article (doi:10.1186/s12934-014-0188-y) contains supplementary material, which is available to authorized users.


Background
Streptavidin (Sav) is a tetrameric protein produced by Streptomyces avidinii, and has tight and specific biotin binding affinity with a dissociation constant of about 10 −15 M [1][2][3]. The Sav-biotin system is widely used for biomolecule labeling, purification, immobilization, and more sophisticated biotechnology applications [4][5][6]. Sav is usually produced using recombinant Escherichia coli carrying the gene encoding the Sav core region (Sav core ) [7][8][9][10]. Sav core consists of the residues Glu-14 to Ala-138 of native full-length Sav (Sav nat ), and is known to have resistance against further degradation by various proteases (Figure 1) [2,11]. The molecular weight of Sav core is 53 kDa, whereas that of Sav nat is 66 kDa. Various Sav variants, such as Sav with higher affinity to biotin, have been produced using Sav core as the base [3,12]; however, there are few reports about the production of Sav nat or its variants, and the N-and C-terminus regions have been recognized as useless parts in Sav.
Streptomyces are gram-positive, filamentous soil bacteria known for their ability to secrete heterologous proteins in culture supernatants [13][14][15]. Streptomyces lividans is the most versatile host among this genus for the production of useful proteins. The secretory production of useful proteins is an important tool for protein production, and is industrially effective due to the simple purification procedures involved without refolding or protein extraction from the cell. There are a large number of reports concerning the secretory production of useful proteins using S. lividans as the host. Pozidis et al. successfully used it to produce a biopharmaceutical, mTNFa, derived from mouse [14]. Zhang et al. reported the secretory production of human interleukin-4 receptor [15]. Thus, S. lividans has great ability to express heterologous proteins, and has attracted attention as an industrial host for protein production [12]. Although the secretory production of various complex proteins, including mammalian proteins, using S. lividans has been demonstrated, there are few reports concerning the secretory production of multimeric proteins, such as dimers or tetramers.
In the present study, we carried out the secretory production of tetrameric Sav nat , which retained affinity to biotin, using recombinant S. lividans as the host. We previously reported the secretory production of various useful proteins using S. lividans as a host [16]. Using this system, we tried to produce Sav nat and successfully demonstrated that S. lividans can be used for the secretory production of active-form tetrameric Sav. This result strongly suggests that S. lividans can be used for the secretory production of other useful multimeric proteins. In addition, to investigate the contribution of the N-and C-terminal regions of Sav nat in Sav production using S. lividans, three truncated Sav variants, Sav ΔN , Sav ΔC , and Sav core , were constructed. Using these variants, we demonstrated that the N-and C-terminal regions of Sav were of significance in Sav production using S. lividans.

Results and discussion
The secretory production of Sav nat using S. lividans and evaluation of biotin binding ability Today, Sav is commercially produced using an E. coli expression system. Although Sav is a secretory-form protein originating from a species of Streptomyces, S. avidinii, its production using recombinant Streptomyces hasn't been demonstrated. In the present report, we carried out secretory production of tetrameric Sav using S. lividans as the host strain. In order to produce Sav as a secretoryform protein using S. lividans, we constructed a vector for Sav expression. Although the synthetic gene of Sav core is usually used for Sav production in E. coli systems, we adopted the original full-length gene of Sav nat derived from S. avidinii, which has high GC-content similar to the genome of S. lividans. Sav nat involves additional amino acid residues at the N-and C-termini compared to Sav core ( Figure 1). Figure 2(A) shows SDS-PAGE analysis of a standard sample of Sav (calculated MW, 53 kDa) and the purified Sav nat produced by S. lividans (calculated MW, 66 kDa). Here, we evaluated biotin binding ability of Sav nat produced by recombinant S. lividans. After immobilization of Sav nat on a biotin-coated polystyrene plate, the unbound molecules were washed out, and then the biotin binding ability was assayed using biotin-HRP. Figure 2(B) shows the assay results for Sav nat produced by S. lividans and a standard sample of Sav. Similar curves were observed between Sav nat produced by S. lividans and the standard sample of Sav, whereas no corresponding curve was found when using BSA as a control. We thus successfully obtained active-form tetrameric Sav nat using the secretory production system of S. lividans.
One of the advantages of secretory production system using S. lividans is the productivity of the protein of interest. There are a lot of reports concerning the secretory production system using S. lividans as the host [13][14][15][16]. The secretory production of useful protein is one of practical ways for industrial protein production due to the simple purification procedures that proteins could be purified directly from the culture broth [14].
Here, to evaluate the utility of our secretory production system in Sav production, the Sav productivity using S. lividans was compared to that of using E. coli. 0.61 mg of Sav Eco was purified from the 100 mL culture broth, whereas the amount of purified Sav nat using S. lividans as the host reached 3.1 mg from the 100 mL culture broth. In order to achieve the further Sav productivity, the signal peptide derived from phospholipase D from S. cinnamoneus (pld signal peptide) was fused in front of the gene encoding Sav nat [16], meaning that Pld signal peptide existed in the N-terminus of the original signal peptide of Sav, and S. lividans/psSav nat was created. As the result, 5.6 mg of Sav nat was purified from the 100 mL culture broth using S. lividans/psSav nat . Thus, the productivity of Sav using S. lividans was 9.2 times higher than that of using E.coli. There are some reports concerning Sav production using E. coli expression system. The amount of purified fulllength Sav from 100 mL of culture broth reached 20 mg [9]. However, in that expression system, fermentor was used for Sav production, and the culture condition needed to be strictly controlled. In this study, Sav and the variants can be expressed and purified by using our simple and conventional procedure. This has a great benefit that we can rapidly produce and evaluate various types of Sav mutants. Recently, Nogueira et al. demonstrated full-length Sav production using the secretory production system using Pichia pastoris [17], and this system may be also applied to Sav variants production. Thus, secretory production can be one of promising tools to produce Sav variants, whereas the large amount of Sav could be obtained using large scale fermentation.

Production of truncated Sav variants and evaluation of the biotin binding ability
There are many reports concerning Sav production using E. coli as the host [7,9,10,18]. In these studies, Sav is produced as Sav core , which consists of the residues Glu-14 to Ala-138 of Sav nat , and Sav core is famous for having resistance to further degradation by many kinds of proteases [2]. However, there are few reports concerning Sav nat production, and the N-and C-terminal regions are not usually regarded as important in Sav production using E. coli. Here, we hypothesized that, in the case of secretory-form Sav production using S. lividans, the N-and C-terminal regions in Sav are significant.
To investigate the role of the N-and C-terminal regions of Sav nat , we tried to construct three Sav variants using S. lividans: Sav core , Sav ΔN , and Sav ΔC (Figure 1). S. lividans/Sav core , S. lividans/Sav ΔN , and S. lividans/Sav ΔC were successfully created. After cultivation, Sav ΔN and Sav ΔC were purified from the culture supernatants of S. lividans transformants; however, not enough Sav core was obtained for purification. Figure 3(A) shows SDS PAGE of purified Sav ΔN , and Sav ΔC using affinity column chromatography. We then evaluated their biotin binding ability. Figure 3(B) shows biotin binding assay results for each Sav variant. Sav ΔN and Sav ΔC showed lower biotin binding ability, compared to Sav nat (Figure 3(B)).
Some secretory proteins derived from Streptomyces are expressed as pro-proteins with a pro-domain. The prodomain is known to play an important role and to promote correct folding and translocation of the protein into the culture supernatant. In secretory production of transglutaminase (MTG), the pro-domain of MTG is a significant factor governing its specific activity and productivity [19]. In the present study, Sav core couldn't be obtained by using S. lividans secretory system although Sav is usually produced as Sav core in E.coli system. In addition, the biotin binding affinity of Sav ΔN and Sav ΔC were decreased, compared to that of Sav nat (Figure 3(B)). Our current research might indicate that the N-and C-terminal regions of Sav are of significance to retain the activity, as well as the pro-domain of MTG.

Evaluation of the thermostability of Sav produced by S. lividans
Sav is a powerful tool in biotechnology and has been applied in various systems such as biomolecule labeling and immobilization of proteins or small molecules [4][5][6]. However, tetrameric Sav can usually be dissociated into monomeric Sav by simple boiling [3,9,12], and thus can be utilized only under mild conditions. Therefore, the construction of thermostable Sav can expand its potential applications. For instance, assembling biomass degradation enzymes from extreme thermophilic microbes on thermostable Sav will allow for artificial cellulosomes capable of reacting with high temperatures [20,21]. There are some reports concerning the importance of the thermostability of Sav or Sav mutants, and Chivers et al. successfully constructed a thermostable Sav mutant with the double mutation S52G/R53D [12].
In this study, we carried out the SDS-PAGE analysis of Sav nat produced by S. lividans after boiling at 100°C for 60 min. Surprisingly, we found thermotolerance in this Sav nat , although tetrameric Sav is generally known to dissociate into monomers and lose its biotin binding affinity after brief boiling. The molecular weight of Sav nat monomer is 16.5 kDa whereas that of Sav core is 13.2 kDa. Figure 4(A) shows the SDS-PAGE analysis of the thermostability of Sav Eco produced by E. coli and Sav nat produced by S. lividans. Each Sav was incubated at 100°C for 60 min. As shown in Figure 4(A), Sav Eco was almost completely dissociated after 5 min of incubation ( Figure 4(A), lanes 1-3), whereas there was no complete dissociation of Sav nat produced by S. lividans (Figure 4(A) lanes 4-6). According to previous reports concerning thermostability of streptavidin, streptavidin-biotin complex indicates high thermostability compared to streptavidin [22,23]. Here, we tried to quantify the amount of biotin binding to Sav nat by HABA assay. However, no biotin was detected in purified Sav nat , and thermostability of couldn't be attributed to biotin binding. We also evaluated the biotin binding ability of each Sav. Figure 4(B) and (C) show the results of assays for biotin binding for each Sav after incubating at 100°C. In the case of Sav Eco produced by E. coli, the biotin binding ability was inactivated after only a 5-min incubation (Figure 4(B)). In the case of Sav nat produced by S. lividans, although its biotin binding ability decreased, it retained much of its binding ability after boiling for 60 min. These results were consistent with those obtained from SDS analysis (Figure 4(A)). Although we also tested the thermostability of Sav ΔN and Sav ΔC , there was no complete dissociation of each Sav variant as well as Sav nat (data not shown). These results may imply that N-and C-terminal regions of Sav nat have no effect of the thermostability.

Conclusion
In the present study, Sav production using S. lividans as a host was successfully achieved. Sav was produced in its tetrameric form and had tight and specific biotin binding affinity. The Sav productivity using S. lividans system was 9.2 fold higher than E. coli system. In secretory production of Sav using S. lividans, the N-and C-terminal regions of Sav were necessary for correct folding and the Sav productivity. These results indicate that our protein secretion system may be used for the secretion of other multimeric proteins. In addition, Sav produced by S. lividans exhibited thermostability, and its biotin binding affinity was retained after boiling. This thermotolerant Sav has many potential applications in biotechnology.

Plasmid construction, transformation, and cultivation
Escherichia coli NovaBlue {endA1 hsdR17(r K12 − m K12 + ) supE44 thi-I gyrA96 relA1 lac recA1/F'[proAB + lacI q ZΔM15::Tn10(Tet r )]} (Novagen, Inc., Madison, WI, USA), used to construct plasmids, was grown in Luria-Bertani (LB) medium containing 40 μg/ml kanamycin at 37°C. The vectors for protein expression using S. lividans as a host were constructed as follows. The strains and the plasmids used in this study are summarized in Table 1. Polymerase chain reaction (PCR) was carried out using PrimeSTAR HS (TAKARA BIO, Shiga, Japan). The gene fragment encoding Sav nat was amplified by PCR using S. avidinii (NBRC13429) as a template with the corresponding primers ( Table 1). The Sav nat fragment was introduced into the NdeI and HindIII sites of pTONA4. The resultant plasmid was called pTONA4-Sav nat . The gene fragment encoding Sav ΔN or Sav ΔC was amplified by PCR using pTONA4-Sav nat as a template with the corresponding primers (Table 1), respectively. The Sav ΔN or Sav ΔC fragments with the signal peptide sequences of Sav, Sav-sig_F, and Sav-sig_R, were introduced into the NdeI and HindIII sites of pTONA4, respectively. The resultant plasmids were called pTONA4-Sav ΔN and pTONA4-Sav ΔC , respectively. The gene fragment encoding Sav core or Sav nat -ps was amplified by PCR using pTONA4-Sav nat as a template with Sav ΔN to ps_F and Sav ΔC _R or Sav_os_to_ps_F and Sav_Rv, respectively ( Table 1). The Sav core or Sav nat -ps fragment was introduced into the NheI and BglII sites of pUC702-pro-sig-term, respectively. The resultant plasmid was called pUC702-ps-Sav core or pUC702-ps-Sav nat , respectively. The gene fragment encoding ps-Sav core or ps-Sav nat was amplified by PCR using pUC702-ps-Sav core or pUC702-ps-Sav nat as a template with ps_F and Sav ΔC _R or ps_F and Sav_R, respectively ( Table 1). The ps-Sav core or ps-Sav nat fragment was introduced into the NdeI and HindIII sites of pTONA4, respectively [24]. The resultant plasmid was called pTONA4-Sav core or pTONA4-ps-Sav nat , respectively. The nucleotide sequences of Sav and Sav variants expressed using S. lividans in this study are shown in Additional file 1. The gene fragment encoding Sav for the construction of the E. coli expression vector was amplified by PCR using pWI3SAFlo318 as a template with KpnI_XhoI_Sav_F and Figure 4 Thermostability of Sav nat produced by using S. lividans in this study was evaluated, and compared to that of Sav Eco produced by using E.coli. (A) SDS-PAGE of Sav Eco produced by E. coli (lanes 1-3) and purified Sav nat produced by S. lividans (lanes 4-6) after boiling at 100°C for 0 min (lanes 1, 4), 10 min (lanes 2, 5), and 60 min (lanes 3, 6). (B) Biotin-avidin conjugation assays of Sav Eco after boiling at 100°C for 0 min (circles), 10 min (squares), and 60 min (diamonds). (C) Biotin-avidin conjugation assays of Sav nat after boiling at 100°C for 0 min (circles), 10 min (squares), and 60 min (diamonds).
Sav_TAG_BamHI_EcoRI_R [25]. The Sav Eco fragment was digested using KpnI and EcoRI and introduced into the KpnI and EcoRI sites of pColdI. The resultant plasmid was called pColdI-Sav Eco .
Hexahistidine tagged Sav produced by E. coli (Sav Eco ) was produced as follows. The plasmid pColdI-Sav was introduced into E. coli BL21 (DE3) pLysS, and the resultant E. coli transformant was called E. coli/Sav Eco . Cells were grown in LB medium to an OD (600 nm) of 0.5 at 37°C, then cells were incubated a further 30 min at 15°C. Expression of the protein was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. After growth for an additional 24 h at 15°C, cells were harvested by centrifugation. The cell pellets were resuspended in 50 mM phosphate, 150 mM NaCl, pH 8.0 and lysed by sonication.

Purification of Sav and Sav variants
Each culture supernatant (300 ml) of S. lividans/Sav nat , S. lividans/Sav core , S. lividans/Sav ΔN , and S. lividans/ Sav ΔC was precipitated by ammonium sulfate. The precipitate of each was collected by centrifugation at 20,000 g for 30 min and dissolved with buffer B (50 mM Tris-HCl, 300 mM NaCl, pH 7.5). Each Sav was purified using TALON metal affinity resins (TAKARA BIO, Shiga, Japan) according to the manufacturer's protocol. After purification, both of the purified protein fractions were dialyzed with 50 mM phosphate buffer and 150 mM NaCl at pH 7.5. In the case of Sav Eco purification, culture broth (600 ml) of cell extract of E. coli/Sav Eco was directly purified using TALON metal affinity resins in a similar fashion. In this study, biotin binding ability was quantified by using Pierce Biotin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA).

Immobilization and biotin-avidin conjugation assays of purified Sav variants
The standard sample of Sav (Nacalai Tesque, Kyoto, Japan) and Sav nat produced by S. lividans were immobilized on biotin coated plates. The maximal amount of each Sav added to the plates was 300 μg/mL. Each Sav was incubated in a 96-well biotin-coated plate (Thermo Fisher Scientific) for 30 min at 4°C. The wells were then washed with TBST three times, and 5 μg/L biotinylated horseradish peroxidase (biotin-HRP) (Thermo Fisher Scientific) per well were incubated for 1 h at room temperature. After washing wells with TBST three times, HRP activity was assayed with an ELISA POD substrate TMB Kit (Nacalai Tesque). Then the absorbance of 450 nm was analyzed with a plate reader (Wallac 1420 ARVOsx).
Sav Eco , Sav ΔN , and Sav ΔC were immobilized on nickelcoated plates (Thermo Fisher Scientific). The maximal amount of each Sav added to the plate was 300 μg/mL. After immobilization of each Sav, HRP activity was assayed in a similar fashion.

Additional file
Additional file 1: The nucleotide sequences of Sav and Sav variants expressed using S. lividans in this study. Underlines and dublet indicate original signal peptides of Sav and signal peptides derived from Streptomyces cinnamoneus phospholipase D, respectively. Italic types show hexahistidine tag. Each nucleotide sequence was introduced into the NdeI and HindIII sites of pTONA4 [23].