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. 2016 Feb;14(2):791-807.
doi: 10.1111/pbi.12430. Epub 2015 Jul 16.

Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Harunipriya Palaniswamy  1 , Divya P Syamaladevi  1   2 , Chakravarthi Mohan  1 , Anna Philip  1 , Anushya Petchiyappan  1 , Subramonian Narayanan  1
Affiliations

Vacuolar targeting of r-proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Harunipriya Palaniswamy et al. Plant Biotechnol J. 2016 Feb.

Abstract

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well-developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high-value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar-targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N-terminal 78-bp-long putative vacuolar-targeting sequence from the N-terminal domain of unknown function (DUF) in Triticum aestivum 6-SFT (sucrose: fructan 6-fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar-targeting determinants at the N-terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His-tagged β-glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar-targeting determinant could localize recombinant proteins (r-proteins) to the vacuole in high concentrations and such targeted r-proteins can be purified from the juice with a few simple steps.

Keywords: GFP; GUS; aprotinin; molecular farming; sugarcane; vacuolar targeting.

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Figures

Figure 1
Figure 1
In silico analysis. (a) Multiple sequence alignment of 6‐SFT and 1‐FFT from monocots. Putative vacuolar‐targeting region (boxed) is flanked by signal peptide cleavage site (red dotted line between G/A and A) and conserved domain (denoted as CD). The domain of unknown function is denoted as DUF as per NCBI annotation. The highly conserved motif FPWSN is highlighted in blue. Ta, Triticum aestivum (Accession No. BAB82469.1); Td, Triticum durum (Accession No. ACH73195.1); As, Aegilops searsii (Accession No. ACH73193.1); At, Aegilops tauschii (Accession No. ACH73194.1); Tu, Triticum urartu (Accession No. ACH73192.1); Hv, Hordeum vulgare (Accession No. CAA03098.1); Bp, Bromus pictus (Accession No. ACN93835.1); Ac, Agropyron cristatum (Accession No. AAK27319.1); Lp, Lolium perenne (Accession No. AAM14603.1); Lt, Lolium temulentum (Accession No. CAD58682.1); Ta, T. aestivum (Accession No. JC7905); Triticum durum (Accession No. ACH73190.1); Tu, Triticum urartu (Accession No. ACH73188.1); As, Aegilops searsii (Accession No. ACH73189.1) (b) Predicted helix–coil–helix–coil secondary structure of 6‐SFT from T. aestivum. (c) Nucleotide and corresponding amino acid sequence of the signal peptide and the putative vacuolar‐targeting region of T. aestivum 6‐SFT. The putative vacuolar‐targeting sequence immediately after the signal peptide cleavage site is underlined and shown in red. The hexapeptide motif (FPWSNE) is shown in blue.
Figure 2
Figure 2
Fluorescent and confocal microscopic images of sugarcane transformed with pSBI‐MF12 containing the 78‐bp vacuolar‐targeting determinant. (a–f) Fluorescent microscopic images. Callus cells showing (a) vacuole (V) with GFP fluorescence, (b) N‐propidium iodide‐stained nucleus and (c) merged images of (a) and (b) distinguishing vacuole and nucleus. (d) Untransformed sugarcane callus. (e) Apoplastic‐targeted GFP fluorescence in the cell wall. (f) Cytosolic‐targeted GFP in the cytoplasm. (g–h) Mature sugarcane parenchyma cells. (g) Untransformed sugarcane. (h) pSBI‐MF12 transgenic sugarcane showing green fluorescence. (i, j) Confocal microscopic images of mature sugarcane stem parenchyma cells. (i) Untransformed sugarcane, (j) pSBI‐MF12 transgenic showing green fluorescence. (k) Fluorescence intensity measured from fluorescent and confocal microscopic images of transformed and untransformed sugarcane stem parenchyma vacuoles. (l–n) Transient GFP expression in celery petioles transformed with pSBI‐MF12 (l) vacuole (V) with GFP fluorescence, (m) N‐propidium iodide‐stained nucleus and (n) merged images of (l) and (m).
Figure 3
Figure 3
(a–c) Fluorescent microscopic image of sugarcane calli transformed with pSBI‐MF13 containing the 18‐bp vacuolar‐targeting determinant. (a) Vacuole (V) with GFP fluorescence, (b) N‐propidium iodide‐stained nucleus and (c) merged images of (a) and (b). (d) GFP expression in apoplast. (e–g) Fluorescent microscopic images of mature sugarcane stem parenchyma cells. (e) Untransformed sugarcane. (f) Localization of GFP in vacuoles of transgenic with pSBI‐MF13. (g) Fluorescence intensity quantified using fluorescent microscopic images of transformed and untransformed sugarcane stem vacuoles. (h–j) Transient expression of GFP in sugar beet transformed with pSBI‐MF13. (h) V (vacuole) with GFP fluorescence. (i) N‐propidium iodide‐stained nucleus (j) Merged images of (h) and (i).
Figure 4
Figure 4
(a) Quantification and comparison of GFP expression in pSBI‐MF10‐ (GFP in cytoplasm), pSBI‐MF11‐ (GFP in apoplast), pSBI‐MF12‐ (GFP in vacuole with VT78) and pSBI‐MF13 (GFP in vacuole with VT18)‐transformed sugarcane juice and leaf by ELISA. UC, untransformed control. Values plotted for GFP accumulation are the mean ± standard error (SE) of replicate plants from each line (n = 10) for pSBI‐MF10 and pSBI‐MF11, (n = 25) for pSBI‐MF12 and (n = 6) for pSBI‐MF13. Means at each time point marked with different letters were significantly different: p1 compared with pSBI‐MF10 juice samples, p2 compared with pSBI‐MF11 juice samples. (b) Spatio‐temporal pattern of GFP accumulation. Means at each time point marked with different letters were significantly different (P = 0.01). (c) Total soluble solids (BRIX) in sugarcane juice. Results shown are the mean ± SE of the replicates from three transgenic lines having high, medium and low GFP.
Figure 5
Figure 5
(a) Southern analysis showing stable integration of VT: GUS in transgenic sugarcane plants. DNA samples were digested with Nco I. Probe DNA of 500 pg is shown in lane 1. Untransformed control and the positive control pSBI‐MF22 plasmid are shown in lanes 2 and 3. Lanes 4–11 show independent transgenic events. (b) Southern analysis showing stable integration of VT: aprotinin in transgenic sugarcane plants. DNA samples were digested with Nco I. Probe DNA of 500 pg is shown in lane 1. Untransformed control and positive control pSBI‐MF32 plasmid are shown in lanes 2 and 3. Lanes 4–11 show independent transgenic events. (c) Crude and dialysed juice of pSBI‐MF22 expressing GUS. Twenty microlitres from untransformed crude sugarcane juice, pSBI‐MF22‐transformed crude sugarcane juice and dialysed sugarcane juice is shown in lanes 2, 3 and 4, respectively. (d) Crude and dialysed juice of pSBI‐MF32 expressing aprotinin. Twenty microlitres the from untransformed crude sugarcane juice, pSBI‐MF32‐transformed crude sugarcane juice and dialysed sugarcane is shown in lanes 2, 3 and 4.
Figure 6
Figure 6
(a–e) Histochemical staining for GUS activity in vacuolar‐targeted GUS transgenics. (a) GUS activity in pSBI‐MF22 (VT:GUS)‐transformed sugarcane juice, (b) plantlets, (c) stem and (d) cross‐section of the stem. (e–h) Histochemical staining for GUS activity in apoplast‐targeted GUS transgenics. (e) GUS activity in pSBI‐MF21 (AT:GUS)‐transformed sugarcane juice, (f) plantlets, (g) stem and (h) cross‐section of the stem. VT:GUS—vacuolar‐targeted GUS; AT:GUS—apoplast‐targeted GUS.
Figure 7
Figure 7
(a) Quantification and comparison of GUS expression in pSBI‐MF20‐ (GUS in cytoplasm), pSBI‐MF21‐ (GUS in apoplast) and pSBI‐MF22 (GUS in vacuole)‐transformed sugarcane juice and leaf extract by fluorometric GUS assay. UC, untransformed control. Values plotted for GUS accumulation are the mean ± standard error (SE) of replicate plants from each line (n = 6). Means at each time point marked with different letters were significantly different: p1 compared with pSBI‐MF20 juice samples, p2 compared with pSBI‐MF21 juice samples. (b) Quantification and comparison of aprotinin expression in pSBI‐MF30‐ (aprotinin in cytoplasm), pSBI‐MF31‐ (aprotinin in apoplast) and pSBI‐MF32 (aprotinin in vacuole)‐transformed sugarcane juice and leaf extract through trypsin inhibition assay. UC, untransformed control.
Figure 8
Figure 8
(a, b) Spatio‐temporal pattern of GUS and aprotinin accumulation and (c, d) total soluble solids (BRIX) in sugarcane juice. The results shown are the mean ± SE of the replicates from three transgenic lines having high, medium and low GUS and aprotinin accumulation. Means at each time point marked with different letters were significantly different at P = 0.01.
Figure 9
Figure 9
Steps in the purification of r‐GUS from pSBI‐MF22‐transformed sugarcane juice. (a) Coomassie Blue‐stained polyacrylamide gel. Twenty microlitres of pSBI‐MF22‐ (lane 1) and pSBI‐MF21 (lane 2)‐transformed sugarcane juice after dialysis, affinity‐purified fraction (lane 3) and desalting column‐purified fraction (lane 4). Four micrograms of lyophilized fraction from pSBI‐MF22 transgenic juice (lane 5) and 4 μg Sigma standard (lane 6). Absence of band in pSBI‐MF21 (GUS in apoplast) lyophilized fraction (lane 7). Molecular weight marker (lane 8) (b) Immunoblot analysis of purified fractions from pSBI‐MF22‐transformed sugarcane juice. Molecular weight marker (lane 1). Absence of band in affinity‐purified fraction from pSBI‐MF21 transgenic juice (lane 2) acts as a control. Affinity‐purified, desalting column‐purified and 4 μg from the lyophilized fraction of pSBI‐MF22 transgenic juice (lanes 3, 4 and 5). Four micrograms of Sigma standard (lane 6) (c) Coomassie Blue‐stained (12%W/V) SDS‐PAGE and immunoblot analysis, demonstrating purity and concentration of recombinant GUS purified from transgenic sugarcane juice through affinity column in comparison with GUS (Sigma). A single band in each lane corresponds to the expected size of the r‐GUS and Sigma GUS. A total of 0.5, 1.5 and 3.0 μg of r‐GUS loaded in lanes 1, 2 and 3; 0.5, 1.5 and 3.0 μg of Sigma GUS loaded in lanes 4, 5 and 6. Lane 7: molecular weight marker. (d) Fluorometric quantification and comparison of r‐GUS and Sigma GUS. Means at each time point marked with different letters were significantly different (P = 0.01).
Figure 10
Figure 10
Steps in the purification of pSBI‐MF31‐ (aprotinin in apoplast) and pSBI‐MF32 (aprotinin in vacuole)‐transformed sugarcane juices. (a) Coomassie Blue‐stained (12%W/V) SDS‐PAGE of pSBI‐MF31‐ and pSBI‐MF32‐transformed sugarcane juices. Twenty microlitres of pSBI‐MF31‐transformed sugarcane juice is shown in lane 1. Twenty microlitres of pSBI‐MF32‐transformed sugarcane juice from the crude sample and after dialysis is shown in lanes 2 and 3. Affinity‐purified, desalting column‐purified and 4 μg of lyophilized fractions from pSBI‐MF32 transgenic juice are shown in lanes 4, 5 and 6. Lane 7 shows molecular weight markers containing known molecular weight proteins. (b) Immunoblot analysis of pSBI‐MF31 and pSBI‐MF32‐transformed sugarcane juices. Affinity‐purified, desalting column‐purified and 4 μg of lyophilized fractions from pSBI‐MF32 transgenic juice are shown in lanes 1, 2 and 3. Lane 4 shows 4 μg of Sigma standard, and lane 5 shows the absence of band in lyophilized fractions from pSBI‐MF31 transgenic juice. Lane 6 shows the molecular weight markers containing known molecular weight proteins. (c) Coomassie Blue‐stained (12%W/V) SDS‐PAGE gel demonstrating purity and concentration of recombinant bovine aprotinin (r‐aprotinin) purified from transgenic sugarcane juice through affinity column in comparison with the bovine aprotinin (Sigma). r‐Aprotinin of 0.5, 1.5, 3.0 μg was provided in lanes 1, 2 and 3,. Bovine aprotinin (Sigma) of 0.5, 1.5 and 3.0 μg was provided in lanes 4, 5 and 6. The absence of band in pSBI‐MF31‐lyophilized fraction is shown in lane 7. Molecular weight marker is shown in lane 8. (d) Comparison and quantification of r‐aprotinin and Sigma aprotinin.
Figure 11
Figure 11
(a) Glycoprotein staining of SDS‐PAGE displaying r‐GUS purified from transgenic sugarcane juice and Sigma GUS. Phytohaemagglutinin, a glycoprotein (Genei), was used as positive control. Molecular weight marker is shown in lane 1. Positive control phytohaemagglutinin (Genei) is shown in lane 2. r‐GUS of 2, 4, 6 μg is shown in lanes 3, 4 and 5. Sigma GUS of 2, 4 and 6 μg is shown in lanes 6, 7 and 8. (b) Coomassie Blue‐stained (12%W/V) SDS‐PAGE showing a single band in each lane, which corresponds to the expected size of r‐GUS and Sigma GUS. Molecular weight marker is shown in lane 1. Positive control phytohaemagglutinin (Genei) is shown in lane 2. r‐GUS of 2, 4 and 6 μg is shown in lanes 3, 4 and 5. Sigma GUS of 2, 4, 6 μg is shown in lanes 6, 7 and 8.
Figure 12
Figure 12
(a) Glycoprotein staining of SDS‐PAGE displaying recombinant bovine aprotinin (r‐aprotinin) purified from transgenic sugarcane juice and bovine aprotinin (Sigma). r‐Aprotinin of 2, 4 and 6 μg is shown in lanes 1, 2 and 3. Bovine aprotinin (Sigma) of 2, 4 and 6 μg is shown in lanes 4, 5 and 6. Phytohaemagglutinin (Genei) as positive control (lane 7). Molecular weight marker (lane 8). (b) Coomassie Blue‐stained SDS‐PAGE gel. r‐Aprotinin of 2, 4 and 6 μg is loaded in lanes 1, 2 and 3. Bovine aprotinin (Sigma) of 2, 4 and 6 μg is shown in lanes 4, 5 and 6. Phytohaemagglutinin (Genei) as positive control (lane 7). Molecular weight marker (lane 8).
Figure 13
Figure 13
(a) Chromatographic profile corresponding to HPLC analysis of r‐GUS and Sigma GUS. The retention time of r‐GUS (green) coincides with that of Sigma standard (pink), indicating the purity of r‐GUS. (b) Fluorometric quantification and comparison of r‐GUS and Sigma GUS. (c) Immunoblot analysis of HPLC‐purified r‐GUS and Sigma GUS. Molecular weight marker is shown in lane 1. HPLC‐purified r‐GUS is in lane 3. HPLC‐purified Sigma GUS is in lane 4. Four micrograms of Sigma standard is loaded in lane 5. (d) Chromatographic profile corresponding to HPLC analysis of r‐aprotinin and Sigma aprotinin. The retention time of r‐aprotinin (green) coincides with that of the Sigma standard (pink), indicating the purity of r‐aprotinin. (e) Quantification and comparison of r‐aprotinin and Sigma aprotinin. (f) Immunoblot analysis of HPLC‐purified r‐aprotinin and Sigma aprotinin. Molecular weight marker is shown in lane 1. HPLC‐purified r‐aprotinin is provided in lane 3. HPLC‐purified Sigma aprotinin is provided in lane 4. Four micrograms of Sigma standard is loaded in lane 5.
Figure 14
Figure 14
Schematic representation of binary vectors used in this study SP, signal peptide; VT78, vacuolar‐targeting sequence 78 bp; VT18, vacuolar‐targeting sequence 18 bp; CI, catalase intron; E, enterokinase sequence; H, His tag.
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