The development and application of new crystallization method for tobacco mosaic virus coat protein

Background Although tobacco mosaic virus (TMV) coat protein (CP) has been isolated from virus particles and its crystals have grown in ammonium sulfate buffers for many years, to date, no one has reported on the crystallization of recombinant TMV-CP connecting peptides expressed in E. coli. Methods In the present papers genetically engineered TMV-CP was expressed, into which hexahistidine (His) tags or glutathione-S-transferase (GST) tags were incorporated. Considering that GST-tags are long peptides and His-tags are short peptides, an attempt was made to grow crystals of TMV-CP cleaved GST-tags (WT-TMV-CP32) and TMV-CP incorporated His-tags (WT-His-TMV-CP12) simultaneously in ammonium sulfate buffers and commercial crystallization reagents. It was found that the 20S disk form of WT-TMV-CP32 and WT-His-TMV-CP12 did not form high resolution crystals by using various crystallization buffers and commercial crystallization reagents. Subsequently, a new experimental method was adopted in which a range of truncated TMV-CP was constructed by removing several amino acids from the N- or the C-terminal, and high resolution crystals were grown in ammonium sulfate buffers and commercial crystallization reagents. Results The new crystallization method was developed and 3.0 Å resolution macromolecular crystal was thereby obtained by removing four amino acids at the C-terminal of His-TMV-CP and connecting six His-tags at the N-terminal of His-TMV-CP (TR-His-TMV-CP19). The Four-layer aggregate disk structure of TR-His-TMV-CP19 was solved. This phenomenon showed that peptides at the C-terminus hindered the growth of high resolution crystals and the peptides interactions at the N-terminus were attributed to the quality of TMV-CP crystals. Conclusion A 3.0 Å resolution macromolecular crystal of TR-His-TMV-CP19 was obtained and the corresponding structure was solved by removing four amino acids at the C-terminus of TMV-CP and connecting His-tags at the N-terminus of TMV-CP. It indicated that short peptides influenced the resolution of TMV-CP crystals.


Background
Tobacco mosaic virus (TMV) has a rod-like appearance and consists of a single, positive strand RNA of 6395 nucleotides encapsulated in a helical virion by approximately 2130 identical coat protein (CP) subunits [1][2][3][4][5][6][7]. CP consists of 158 amino acids that were assembled into four main alpha-helices joined by a prominent loop proximal to the axis of the virion [8][9][10][11][12][13][14][15][16]. TMV-CP played an important role in the self-assembly of TMV through an initial RNA recognition reaction that triggers the assembly, it was believed to be necessary for virus assembly initiation and elongation [8,[17][18][19][20][21][22][23][24]. The biological physical properties of TMV-CP were often determined by the structure of TMV. The TMV structure reported in 1986 was studied based on an electron density map at 3.6 Å resolution by X-ray fiber diffraction [9], and then this structure of the complete virus was determined at 2.9 Å resolution by X-ray fiber diffraction methods [25]. TMV-CP assembly systems, consisting of 34-subunit aggregate of TMV-CP crystallized as a dimer of bilayer disks having 17 subunits per layer, were crystallized and solved at 2.8 Å resolution [2]. The crystalline structure of the Four-layer aggregate of TMV-CP was determined at 2.4 Å resolution by using X-ray diffraction from crystals maintained at cryogenic temperatures. This structure emphasized the importance of water in biological macromolecular assemblies [22,23]. The circular permutants of TMV-CP were crystallized and solved by molecular replacement at 3.0 Å resolution by using X-ray diffraction [26]. The structure of TMV was also obtained by using high resolution transmission electron microscopy [27][28][29].
TMV-CP was usually propagated and isolated from Nicotiana tabacum (N. tabacum) infected by TMV, and TMV-CP existed as a number of aggregates, depending on PH, ionic strength, temperature, protein concentration, and other factors [5,12,[14][15][16][17][18][19][20][21]. At 0.1 mol/L ionic strength orthophosphate solution and pH equal to or greater than 8.0, TMV-CP existed as protein A or 4S protein (a dynamic equilibrium between monomers, trimers, and pentamers of TMV-CP). At 0.1 mol/L ionic strength orthophosphate solution and pH near 7.0, TMV-CP was transformed into the 20S aggregate form (disks consisting of 34 monomers, also named as the 20S structure) with an admixture of 4S protein [30][31][32][33]. At 0.1 mol/L ionic strength orthophosphate solution and pH equal to or less than 6.0, TMV-CP was completely The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. (C) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

Figure 2
Alignment of the TMV-CP Sequences, the identical residues were marked below by an asterisk.
In the present investigation, the genetically engineered structure of TMV-CP was the concern: a series of the recombinant expression vectors contained TMV-CP genes were constructed and transformed into E.coli, and the recombinant protein of TMV-CP were expressed, and 3.0 Å resolution TR-His-TMV-CP 19 (incorporated Histags at the N-terminus of TMV-CP and truncated four amino acids at the C-terminus of TMV-CP) macromolecular crystals were obtained.

Results and Discussions
Identification to Recombinant Vectors TMV-RNA has been isolated from TMV particles (propagated in N. tabacum K 326 ) and reverse transcribed into cDNA by primer cDNA and reverse transcriptase (TaKaRa). The genetic fragment of wild type TMV-CP (WT-GST-TMV-CP 32 , with the restriction enzymes of BamH I/Xho I; WT-His-TMV-CP 12 , with the restriction enzymes of Nde I/Xho I; ) were amplified by using cDNA as template. The following series of genetic fragments, four amino acids truncated at the C-terminus of WT-His-TMV-CP 12 , (TR-His-TMV-CP 19 ), three amino acids truncated at the N-terminus of WT-His-TMV-CP 12 and four amino acids truncated at the C-terminus of WT-His-TMV-CP 12 (TR-His-TMV-CP 62 ), three amino acids truncated at the N-terminus of WT-His-TMV-CP 12 , and five amino acids truncated at the C-terminus of WT-His-TMV-CP 12 (TR-His-TMV-CP 68 ), were also amplified by PCR. Compared with the DNA marker, all the PCR products migrated as expected at approximately 500 bp, and the PCR products were marked as the WT-GST-TMV-CP 32  The corresponding clones were sequenced by ABI Automatic DNA Sequence Machine, and the correct sequences were obtained and aligned ( Figure 2). The DNA sequences of WT-GST-TMV-CP 32 , WT-His-TMV-CP 12 , TR-His-TMV-CP 19 , TR-His-TMV-CP 62 , and TR-His-TMV-CP 68 were similar that of WT-TMV-CP (isolated from TMV), except for the presence of short peptides incorporated at the N-terminal of TMV-CP in their DNA sequences. These correct proteins were successfully cloned to the expression host, E.coli BL 21 (DE 3 )-RIL (TakaRa), for protein expression.

Confirmation of the Proteins Expressed and Purified by Gel Filtration
Expressed proteins of WT-GST-TMV-CP 32 , WT-His-TMV-CP 12 , TR-His-TMV-CP 19 , TR-His-TMV-CP 62 , and TR-His-TMV-CP 68 were initially assayed by Coomassie brilliant blue method in a small scale experiment in which the final volume was 10 mL( Figure 3A).The protein products including the whole cell lysates and the target proteins were confirmed by 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The molecular mass of WT-GST-TMV-CP 32 was test by migration at approximately 43.5 kDa ( Figure 3B); the molecular mass of WT-His-TMV-CP 12 ( Figure 3C), Results showed that increased salt and ionic strength increased by the crystallization reagent changed the crystallization from octahedral crystals to bar and lamellar crystals. (I) WT-TMV-CP 32 microcrystal that was cloned using the vector of PGEX-6P-1, purified using His-tags, cleaved GST-tags by PreScission Protease. In addition, seeding tools were used and crystallization reagents were changed, including the crystallization reagents of Hampton research, in an attempt to improve the quality and size of the crystals or to produce a different crystal form. Only twin crystals or polycrystalline were obtained, as shown in (J), (K), and (L). Figure 3D), TR-His-TMV-CP 62 , and TR-His-TMV-CP 68 were test by in the same migration approximately at 18.5 kDa. The molecular mass of WT-TMV-CP 32 (WT-GST-TMV-CP 32 cleaved GSTtags) was test by migration at approximately 17.5 kDa ( Figure 3E), and the molecular mass of TR-His-TMV-CP 12 ( Figure 3F) and TR-His-TMV-CP 19 ( Figure 3G) were test by in the same migration at approximately 18.5 kDa.

Disk state of TR-His-TMV-CP 19 in Solution
In the size exclusion chromatography, the retention volume of N-His-TMV-CP 19 protein under 20 mM Sodium Phosphate buffer and 100 mM Sodium Chloride solution (PH 8.0) was the oligomeric state (dimmers and monomers), and the retention volume of TR-His-TMV-CP 19 was transformed to disks mostly after dialyzing against 0.2-0.3 mol/L ammonium sulfate and 0.1 mol/L Tris ( PH 8.0 ) solution at room temperature for more than 10 hr. The disk state of TR-His-TMV-CP 19 was confirmed by the map of SEC ( Figure 6A) and Native-PAGE simultaneously ( Figure 6B and Figure 6C).

Identification and Diffraction of Crystals
Macromolecular crystals were grown in crystallization buffers with high levels of supersaturation, often reaching several hundred percent. WT-TMV-CP 32 , WT-His-TMV-CP 12 , TR-His-TMV-CP 62 , TR-His-TMV-CP 68 crystals ( Figure 4) were cultured by using Index Screen (Hampton research) and ammonium sulfate buffers. These crystals were optimized by seeding method, but high resolution crystals were not obtained.
On the contrary, TR-His-TMV-CP 19 crystals ( Figure 5) (protein concentration: 14 mg/mL) with 3.0 Å resolution were obtained in 0.25 mol/L ammonium sulfate and 0.1 mol/L Tris solution at 295 K, PH 7.7 ( Figure 6), and the Four-layer aggregate disk structure of TR-His-TMV-CP 19 was solved.
TMV-CP has been available as a recombinant protein expressed in E.coli for more than 20 years [37,38]. The incorporation of His-tags at the C-terminal of TMV-CP has been reported recently by introducing His-tags into TMV-CP to facilitate their purification [39]. To date, however, no one has reported on the crystallization of recombinant TMV-CP connecting peptides expressed in E. coli, except on the residues of chemical modification of TMV-CP [26,[40][41][42][43]. These recombinant proteins connecting with peptides often did not affect the biological activity of the engineered proteins, located at the exterior of the TMV-CP disks. To obtain high resolution crystals, the expression vectors containing TMV-CP Figure 5 Examples of TR-His-TMV-CP 19 crystals with fused short peptides and truncated four amino acids from the C-terminus (Showed in Table 4), the scale bar represents 0.1 mm.  Figure 5B, (E) X-ray crystal diffraction of TR-His-TMV-CP19 marked in Figure  6A, (F) X-ray crystal diffraction of TR-His-TMV-CP19 marked in Figure 6G, (G) X-ray crystal diffraction of TR-His-TMV-CP19 marked in Figure 6J, (H) X-ray crystal diffraction of TR-His-TMV-CP19 obtained from the conditions of Figure 6J, (I) The Four-layer aggregate structure of TR-His-TMV-CP19 incorporated His-tags. fragments were first constructed and expressed. An attempt was made to harvest the WT-His-TMV-CP 12 fragments by using thrombin cleavage to cleave His-tags. His-tags were not cleaved when the proportion of thrombin cleavage and recombinant proteins was increased from 1:1 to 8:1. Subsequently, another genetically engineered WT-GST-TMV-CP 32 was constructed and expressed. Compared with His-tags, the GST-tags were easily cleaved by PreScission Protease. The crystals of WT-TMV-CP 32 and WT-His-TMV-CP 12 were grown in ammonium sulfate buffers and commercial crystallization reagents simultaneously. No high resolution crystals were formed in the hanging drops when the proportion of WT-TMV-CP 32 to crystallization solution was 1:1. Only tiny octahedral WT-His-TMV-CP 12 crystals were grown in the crystallization room at 295 K. No matter how long the growth time was, the crystals did not grow bigger in the crystallization buffers and commercial crystallization reagents. To maintain the physical properties of WT-His-TMV-CP 12 Figure 6A).
The crystals of TR-His-TMV-CP 19 were obtained at 2.9-7.0 Å resolution ( Figure 6E-6H) and the crystal of WT-His-TMV-CP 12 was obtained without diffraction. A Four-layer aggregate crystal structure of TR-His-TMV-CP 19 was obtained by removing four amino acids at the C-terminal of His-TMV-CP and connecting short   The WT-His-TMV-CP 12 fragment was amplified by using primer 1 and primer 2 . b The WT-GST-TMV-CP 32 fragment was amplified by using primer 3 and primer 2 .
c The TR-His-TMV-CP 19 fragment was amplified by using primer 1 and primer 9 . d The TR-His-TMV-CP 62 fragment was amplified by using primer 6 and primer 2 . The TR-His-TMV-CP 68 fragment was amplified by using primer 6 and primer 8 .
peptides at the N-terminal of His-TMV-CP (TR-His-TMV-CP 19 ). A diagram of the growth curve of TR-His-TMV-CP 19 crystallization was drawn ( Figure 7). Compared with the crystals of WT-TMV-CP (isolated from TMV particles), the Propagation, purification and acquisition the macromolecular crystals of TR-His-TMV-CP 19 from E.coli were very facile process. By investigating, it was found that the crystals of WT-His-TMV-CP 12 had close relationships with protein concentration, ionic strength and PH of the solution. After truncating four amino acids at the C-terminal of WT-His-TMV-CP 12 , a Four-layer aggregate structure of TR-His-TMV-CP 19 was determined at 3.0 Å resolution by using the technique of Hanging-drop vapor diffusion and seeding methods, but the high resolution crystals of WT-TMV-CP 32 , WT-His-TMV-CP 12 , TR-His-TMV-CP 62 , and TR-His-TMV-CP 68 did not obtain.

Conclusions
The     vapor at 277 and 295 K. Seeding methods were performed at 295 K after 1-7 d, the good macromolecular crystals appeared under different conditions. 2.9-7.0 Å resolution of TR-His-TMV-CP 19 (concentration 14 mg/mL) macromolecular crystals were obtained in crystallization pool solution consisting of 0.25 mol/L ammonium sulfate and 0.1 mol/L Tris at pH 7.7. Then, using the same method, the crystals of WT-His-TMV-CP 12 without resolution and some tiny WT-TMV-CP 32 crystals, were also obtained by series of seeding experiments. It was showed from the experiment that: the genetically engineered proteins of TR-His-TMV-CP 19 could grow high resolution crystals. Hence, the present investigations suggest that the C-terminal of TMV-CP was unstable for crystallization buffer, and the amino acids at the Cterminus were hypothesized to be very flexible. Additionally, the inserted sites of short peptides of TMV-CP could access the grown crystals. Short peptides have a positive influence on the stability of the biophysical properties of TMV-CP. Compared with WT-TMV-CP isolated from TMV particles, this recombinant protein of TR-His-TMV-CP 19 is easy to purify and grow crystals. Thus, the latter can be applied to structural biology and structure-based drug design.

Methods
TMV (common strain) was isolated from N. tabacum K 326 leaves infected by TMV, which were cultivated in the Greenhouse of Center for Research and Development of Fine Chemicals of Guizhou University, and purified by the method described by Gooding [44], and modified by Shire [16,45]. TMV-CP was prepared by Scheel [18]. TMV-RNA was extracted from purified virus by treating with phenol and SDS [46][47][48]. In order to obtain the generation of full-length viral cDNA sequence, TMV-RNA was reverse transcribed using primer cDNA (Table 1) in 50 Table 4 Examples of TR-His-TMV-CP 19 crystals in Figure 5 Examples Protein Concentration Crystal Conditions Crystal appearance Resolutions  mmol/L Tris at pH 8.0, 8.0 mmol/L magnesium chloride, 75 mmol/L potassium chloride, 10 mmol/L DL-Dithiothreitol, 1.0 mmol/L dNTPs, 0.5 unit/μL AMV reverse transcriptase (TaKaRa), and 1.0 unit/μL RNase inhibitor (TaKaRa) for 1.5 hr at 315 K.
The dsDNA of correct length was purified and identified by 1% agarose gel electrophoresis. Both plasmid pET28a (Novagen) and CP were digested with Nde I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in pET28a (pET28a-WT-His-TMV-CP 12 , pET28a-TR-His-TMV-CP 19 , pET28a-TR-His-TMV-CP 62 , and pET28a-TR-His-TMV-CP 68 ). Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP 32 ). Transcription reactions were performed by using the corresponding transcription system. E. coli BL 21 (DE 3 )-RIL (TaKaRa) cultures were transformed into vectors involving aforementioned recombinant plasmid. Expression plasmids were grown in Luria-Bertani (LB) medium containing 30 μg/mL kanamycin at 310 K until the OD 600 reached 0.65-1.0. After cooling the cultures to 289 K, the expression product was induced by the addition of 1.0 mmol/L IPTG, and the culture was incubated for 16 hr. The cells were harvested by centrifugation and resuspended in 35 mL lysis buffer (100 mmol/L sodium chloride, 50 mmol/L phosphate buffer, pH 8.0, 10 mmol/L ß-mercaptoethanol). Then, the cells were thawed, lysed by supersonic device, and then centrifuged at 15000 rpm for 30 min at 277 K. The supernate was then passed through 0.22 mm syringe filters (Millipore) and loaded onto a Ni Sepharose High performance column (GE Healthcare, 5mL), washed with five column volumes of 40 mmol/L imidazole, and eluted with 400 mmol/L imidazole. The N-terminal His-tags failed to cleaved with thrombin (1.0 unit/mg) and N-terminal GST-tags was cleaved successfully with PreScission Protease (1.0 unit/mg) by incubating overnight at 277 K. The cleaved GST-tags and uncleaved His-tags were removed by the same chelating column, and the flow-through was concentrated in an Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa filter and then loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated in the dialysis solution (20 mmol/L orthophosphate and 100 mmol/L sodium chloride, pH 8.0). The protein was concentrated to 5.0-25 mg/mLfor the crystallization trials by using Amicon Ultra centrifugal filter units (Millipore) with a 10 kDa molecular weight cutoff. The target proteins were briefly stored at 277 K.
The purification proteins were dialyzed against the appropriate high-salt solution at room temperature to obtain the Four-layer aggregate (20S disk) [35,36,[49][50][51][52]. The 20S disk form proteins were confirmed by Size Exclusion Chromatography (SEC) and Native-polyacrylamide gel Electrophoresis (Native-PAGE) method. SEC was performed at room temperature by using a calibrated Superdex 200 10/300 GL column (GE Healthcare) attached to an AKTApurifier fast protein liquid chromatography system (GE Healthcare). The column was equilibrated with a solution containing 20 mM orthophosphate (pH 8.0), 100 mM NaCl solution. Molecular mass standards (Bio-Rad) used are: Thyroglobulin (669 kDa), Ferritin (440 kDa), BSA (67 kDa), β-lactoglobulin (35 kDa), Ribonuclease A (13.7 kDa), Cytochrome (13.6 kDa), Aprotinin (6.51 kDa) and Vitamin B12 (1.36 kDa). Protein was monitored by absorbance at the wavelength of 280 nm. The crystals of purified proteins were obtained by the technique of Hanging-drop vapor diffusion. The protein concentration was 5.0-25 mg/mL, and the crystallization solutions contained 0.1-0.3 mol/L ammonium sulfate and 0.1 mol/L Tris at PH 6.5-8.5 (Table 3) for 1-7 d at 293-298 K. The crystals (Table 4) were first soaked with cryoprotection (the reservoir solution containing an extra 30% glycerol), and then mounted and flash-frozen in liquid nitrogen [33,34]. Diffraction data were collected Table 5 Data collection and refinement statistics of TR-His-TMV-CP 19 crystals in Figure 6I TR-His-TMV CP 19 Data collection