Development of Series of Affinity Tags in Streptomyces

Streptomyces are of great biological and industrial significance due to their complex morphological development and ability to produce numerous secondary metabolites. However, the intrinsic biochemical mechanisms underlying morphogenesis and secondary metabolism are rarely revealed, partially because of the limited availability of the biochemical tools in Streptomyces. Here we provided series of integrative vectors with various affinity tags, including single tags 3×FLAG, 3×HA, 3×Strep-tag II, 18×His, 13×Myc, and dual tags, all of which were driven from a strong constitutive promoter ermEp*. Using a sigma factor SigT from S. coelicolor as a model, we successfully expressed and immuno-detected SigT fused with all tags. Moreover, after SigT was N-terminally tagged with 3×FLAG and C-terminally tagged with 18×His, we isolated SigT-interactive proteins from the S. coelicolor lysate based on the tandem affinity purification (TAP). Particularly, among the proteins purified, the SigT cognate anti-sigma factor RstA ranked the top with the most total independent spectra. These data suggested the feasibility of these affinity tags in Streptomyces, which will be widely employed to explore the biochemical mechanisms to further understand the dynamic and elaborate regulation in this genus.

In Streptomyces, limited tags have been successfully used, such as FLAG in ChIP assays 10,20 , eGFP for protein degradation monitoring [21][22][23] , and both tags for protein-protein interaction assays 22 . eGFP and mCherry have tremendously facilitated visualization of dynamic localization of morphogenesis-related proteins, revealing the subtle morphological changes and authentic protein functions [24][25][26] . However, these bulky fluorescent proteins might cause steric hindrance to impede the appropriate protein folding 27 and block the interaction with others 28 .
Here we provided series of Streptomyces vectors containing various codon-optimized small single tag or dual tags, including 3×FLAG, 3×HA, 13×c-Myc, 18×His and 3×Strep-tag II. All these tags have been successfully expressed in fusion with the sigma factor SigT in S. coelicolor, and tandem affinity purification (TAP) was demonstrated for the first time in Streptomyces to show the feasibility of these tags without functional disruption of the fused proteins.

Results
Construction of Streptomyces vectors with affinity tags. Antibodies are powerful tools in investigating protein functions nowadays. Both protein-specific polyclonal and monoclonal antibodies against endogenously expressed PhoP and ScbR2 have been reported in Streptomyces ChIP assays 16,29 . However, though polyclonal antibodies can be readily raised, quality control for affinity and specificity is problematic, and preparation of monoclonal antibodies will be time-consuming and risky 30,31 . One of resolutions to overcome these problems is to express the target proteins in fusion with commonly used small epitopes, such as 6×Histidine, 8-amino acid FLAG tag (DYKDDDDK), 9-amino acid influenza hemagglutinin (HA) tag (YPYDVPDYA), 10-amino acid c-Myc tag (EQKLISEEDL), etc 32 . The 8-amino acid Strep-tag II (WSHPQFEK) is a recently developed epitope exhibiting intrinsic affinity toward streptavidin, and can be used to efficiently purify fusion proteins by Strep-Tactin conjugated beads 33 . Fusion of one of these small tags to the target proteins has been proven to have the minimal influence on their native conformation and functions 34 . Moreover, there are commercially available monoclonal antibodies against these affinity tags for various purposes, such as immunoblot, immunoprecipitation (IP), immunohistochemistry (IHC) and ChIP, with the advantages of predominantly high affinity, specificity, and time-and labor-saving.
To expand their applications and increase their antigenicity in Streptomyces, multiple tandems of these small epitopes were generated after codon-optimization. The 22-amino acid 3×FLAG (DYKDHDGDYKDHDIDYKDDDDK), and tandemly arrayed small tags including 3×HA, 13×Myc, 18×His, 3× Strep-tag II were all expressed under the control of a strong constitutive promoter ermEp* 22 . Eight vectors (pSN1-pSN8) ( Fig. 1) were the derives of the integrative promoter-probing vehicle pIJ8660 35 , where ermEp* was inserted between two BglII sites to remove the multiple cloning sites (MCS) on the parental vector, and the egfp reporter gene was replaced by the synthetic DNA fragments encoding the tandem epitopes together with the redesigned MCS. To meet the demand of labeling proteins at alternative ends, the 18×His tag was positioned at the C-terminus of the fusion proteins, while others were N-terminally tagged. Meanwhile, for the purpose of tandem affinity purification (TAP), two vectors were constructed by combination of N-terminal 3×FLAG or 3× Strep-tag II with C-terminal 18×His, given that 18×His-tagged fusion proteins can be purified both at native and denatured conditions, and the purification matrix Ni 2+ -NTA is relatively inexpensive, while anti-FLAG beads and Strep-Tactin beads are in excellent quality control in protein purification and immunoprecipitation 10,33 . Moreover, to avoid possible steric hindrance from the tandem epitopes, a linker sequence 10×Gly was placed between 3×HA, 3×FLAG, or 3×Strep-tag II and the target protein, while a linker sequence GGSGGGGGG was placed between the target protein and 18×His ( Fig. 1).

Expression of fusion proteins with affinity tags.
To validate these affinity tags in Streptomyces, an extracytoplasmic functions (ECF) sigma factor SigT from S. coelicolor was expressed from all above vectors. SigT can be physically protected by its cognate anti-sigma factor RstA from degradation, and is involved in dual positive feedback regulation of morphogenesis and secondary metabolism of S. coelicolor. We have previously tagged it with 3×FLAG and eGFP for immuno-detection and immunoprecipitation 22,23 . However, a 10-amino acid flexible linker rich in Pro and Gly was essential for the proper expression of SigT-GFP 22 , suggesting that GFP, which is 236-amino acid long, might interfere with the proper conformation of SigT.
sigT coding sequence was cloned in all constructs in fusion with tags, and expressed in S. coelicolor. Immuno-blot assays showed SigT fusion proteins were successfully expressed in Streptomyces with α-FLAG, HA, Myc, His and Strep-tag II antibodies, respectively (Fig. 2). All the antibodies specifically recognized the corresponding epitopes, since only the SigT fusion protein bands were clearly observed in the immuno-blots. This highly reduced cross-reactivity will guarantee the exclusion of the false positive in downstream assays, especially such as ChIP. All the data suggested that these tags could be readily expressed and fused with Streptomyces proteins.

Identification of SigT-interactive proteins by tandem affinity purification (TAP). Previous report
has shown that an artificial extension of target proteins with 3×FLAG tag will not disturb the functionality in vivo by ChIP assays in studying protein-DNA interaction 20 . Here we further validated the feasibility of these affinity tags in the tandem affinity purification (TAP), which is a powerful tool in exploring the protein-protein interaction in vivo 36 . SigT has been shown subject to protein degradation during the secondary metabolism of S. coelicolor 23 , but its degradation was blocked in the proteasome-deficient ΔprcB/A mutant 21 , suggesting that SigT remain intact while interacting with other proteins involved in its degradation in this mutant. Therefore the TAP assay was demonstrated with SigT as a model in the ΔprcB/A mutant to explore its potential interactome (Fig. 3). SigT was expressed in pSN7 with dual tags 3×FLAG at its N-terminus and 18×His at C-terminus. 3FLAG-SigT-18His could be immuno-detected in the ΔprcB/A mutant (Fig. 3, lane 1). Though some SigT fusion protein flowed away after loading on Ni 2+ -NTA, initial affinity binding to nickel could capture most SigT protein (Fig. 3, lane   Figure 2. Western blot analysis of expression of SigT fused with series affinity tags in S. coelicolor M145 from pSN2-pSN8. All samples were immuno-blotted with tag-specific antibodies, and coomassie blue staining of total protein served as a loading control.

2-7)
. Then the eluent was reloaded on the anti-FLAG M2 monoclonal antibody-conjugated agarose. After careful washes to a low background, SigT together with its interactive-proteins dissociated from the beads by heat denature (Fig. 3, lane 8-12). After buffer exchange and trypsin digestion, the final eluent was subject to HPLC-MS/MS for peptide sequencing and identification.
Totally 28 proteins were identified in this TAP assay (Table 1) (see Supplementary data for the detailed information). Among them, SigT ranked No.2 with 100% group probability and showed 3 independent sequenced peptides, consistent with this protein as a bait in TAP. Interestingly, the anti-sigma factor RstA (SCO3891) had the most total independent spectra, and similarly, three peptides were independently sequenced as SigT, while all other 26 proteins only had one matched peptide. These data were consistent with our previous immunoprecipitation assay that SigT could physically interact with RstA 22 . Moreover, other proteins potentially involved in biosynthesis, regulation, transportation, etc, together with several proteins with unknown functions were also identified, suggesting that SigT might participate in diverse physiological processes by protein-protein interaction. All these data suggested that our dual tag strategy could be applied in TAP to study protein-protein interaction in Streptomyces.

Discussion
The filamentous bacteria Streptomyces are becoming the focus for its industrial values to produce numerous secondary metabolites in medicinal and agricultural applications. High through-put genome sequencing also showed a large biosynthetic gene cluster pool in this genus, suggesting a huge capacity for natural product production. Many genetic tools and in vitro assays have been established to reveal the genetic circuits and pathway cross-talks controlling the physiology of Streptomyces. Here we provided biochemical approaches with several affinity tags for in vivo protein expression and detection, and also for the first time established tandem affinity purification (TAP) in Streptomyces to study protein-protein interaction with SigT as a bait. These in vivo biochemical tools can also be expanded to explore the real-time dynamics of protein-DNA interaction (such as ChIP), to establish the protein-protein interaction network (or interactome), and to examine protein-protein competition or coordination in regulating gene expression on the promoters of regulons, etc. All these mechanisms in Streptomyces will be the basis of building up biological models of their morphological development and rational designing of biosynthetic and regulatory circuits to exploit their potentialities in secondary metabolite production to the utmost.

Materials and Methods
Strains and media. Streptomyces coelicolor wild type M145 and the ΔprcB/A mutant 21 were used for protein expression in vivo. Escherichia coli strain TG1 was a host for the routine plasmid sub-cloning. E. coli strain ET12456 containing pUZ8002 was used for conjugation of plasmids from E. coli to Streptomyces 8 .
All E. coli cells were cultured in LB medium. The inter-species conjugations were demonstrated according to the protocol described on the SFM medium 8 . For SigT fusion protein expression, all Streptomyces strains were cultured in TSB supplemented with 5% PEG6000 (TSBP) to the logarithmic stage. For the TAP assay, the S. coelicolor ΔprcB/A mutant expressing 3FLAG-SigT-18His was initially cultured in TSBP for 1 day, and then transferred to the liquid R5-medium for the secondary metabolism development after continuous culture for 2 days 21 .

Plasmid construction. All the plasmids and primers in this study were listed in Supplementary data
Tables S1 and S2, respectively. Fragments I, II, III and IV containing NdeI-3HA-linker-MCS-TAA-NotI, NdeI-3FLAG-linker-MCS-linker-18His-TGA-NotI, NdeI-3Strep-tag II-linker-MCS-TAA-NotI and NdeI-MCS-13Myc-TAA-NotI (herein TAA and TGA encode the stop codons, and MCS is multiple cloning sites), respectively, were synthesized with optimized-codons for Streptomyces in Qinglan company, China (see Supplementary data for detailed sequences). ermEp* was amplified with primers 1 and 2 from pIJ8630-ermEp* 22 , digested with BamHI, and inserted into the BglII site of pIJ8660 35 to create the vector pIJ8660-ermEp*. Then the egfp gene was replaced with fragments I-IV at the NdeI/NotI site to give rise to plasmids pSN2, 7, 4 and 5, respectively. BglII/EcoRI fragments from pSN2 and pSN7 were cloned into BglII/EcoRI-digested pSN5 to create plasmids pSN1 and pSN6, respectively. Then pSN1 was digested by BglII and EcoRI, and the smaller DNA fragment was gel-recovered and inserted into BglII/EcoRI-digested pSN7 to make plasmid pSN3. BglII/EcoRI fragment of pSN4 were replaced with BglII/EcoRI fragment from pSN7 to produce plasmid pSN8. sigT was amplified from the genomic DNA of M145 with primers 3 and 4, digested with BglII and XbaI, and ligated into the BglII/XbaI site of pSN2 to pSN8 to create sigT expression plasmids as listed in Table S1. Total protein from S. coelicolor ΔprcB/A expressing 3FLAG-SigT-18His was subject to sequential nickel-based and anti-FLAG antibody-based affinity purification. Samples were taken during TAP as described in Materials and Methods. L: total lysate. FT: flow through. W: wash. E: elution.
Tandem affinity purification (TAP) and protein identification. The total protein was prepared from mycelia of the ΔprcB/A mutant expressing 3FLAG-SigT-18His in buffer A (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM imidazole). Initial nickel-based purification was described by the manufacture (Merck). Briefly, the lysate was loaded on the buffer A-equilibrated Ni 2+ -NTA (the flow-through as FT), and incubated for 2 hours at 4 °C. After two rounds of washing with 10-column volumes of buffer A (W1, W2), the binding proteins were eluted with buffer B (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5% glycerol, 500 mM imidazole) for 6 times, each with 500 μl of buffer B (E1-E6). All the eluents were combined and EDTA was added to a final concentration of 1 mM. Then the protein mixture was incubated with 250 μl of anti-FLAG M2 agarose gel (Sigma) equilibrated with buffer C (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 2 hours at 4 °C. After a short centrifugation (500 rpm, 3 min), the supernatant (FT) was discarded and the pellet was washed 3 times with 1 ml of buffer C (W1). After centrifugation at 500 rpm for 3 min, the supernatant was discarded as much as possible. 500 μl of 20 mM Tris-HCl (pH 8.0) buffer was added to the pellet and boiled for 5 min for protein elution, and this process was repeated for 2 times (E1-E3). All the eluents were combined and the purified proteins were digested with trypsin and identified by HPLC/MS/MS as described before 37 .