Designing Primers with a Plant Signal Peptide to Enhance the Expression of GBA1 in Transgenic Soybean Plants

Transgenic plants offer advantages for the manufacture of recombinant proteins with terminal mannose residues on their glycan chains. So plants are chosen as source of pharmaceutical products and for the development of alternative expression systems to produce recombinant lysosomal enzymes. In the present study the sequence of the natural cDNA encoding for the human lysosomal enzyme glucocerebrosidase (GCD) was modified to enhance its expression in soybean plants. The glucocerebrosidase gene signal peptide was substituted with that signal peptide for the Arabidopsis thaliana basic endochitinase gene to support the co-translational translocation into the endoplasmic reticulum (ER), and the storage vacuole. So, targeting signal from tobacco chitinase A, to facilitate GCD trafficking from the ER to the storage vacuole, appropriate primers were designed containing both an ER and vacuolar targeting signals, (VTS). Those primers were used for PCR amplification of the human GBA gene (Hu-GBA) gene from constructed PGEM-GBA plasmid which was cloned in the plant expression vector pCAMBIA1304. The resulted construct was transported in Agrobacterium tumefaciens strain LBA4404 and was used for transformation of cotyledon explants. After 5-day of seedling, cotyledons were cut and used as explants. After infection and co-cultivation, hygromycin B was added in selection media as a selective agent for the transformants cotyledons. The presence of the Hu-GBA transgene in the genomes of transgenic plants was determined by polymerase chain reaction PCR as a band of size1587 bp. The GBA mRNA expression in modified soybean was detected by qRT-PCR compared with control GBA mRNA.


Introduction:
The concept of utilizing plants for the production of valuable pharmaceuticals, such as vaccines and recombinant proteins, was introduced over twenty years ago (1). Compared with bacteria, yeast, mammalian and insect cell culture expression systems, plants have many advantages for the production of "recombinant proteins" such as the very low production costs, safety, very high scaleup potential, and an initial decrease investment requirements (2). Molecular cultivation refers to the usage of plants and cell cultures to produce "recombinant proteins". Glycosylation is a major factor when attempting to produce recombinant proteins using Escherichia coli based expression system. Glycosylation is often requisite for many proteins to be folded properly, and can play an important role in protein stability and turnover rates (3). In additional non-native glycan structures can be immunogenic. However, recombinant proteins in plant will be modified when targeted for retention within the "endoplasmic reticulum (ER)" by the addition of high-mannose-type N-glycans (4). The immunogenic complex N-glycans and O-glycans are not added until later in the secretory pathway. Several studies have shown that recombinant proteins will be retained within the ER when they are modified by high-mannose-type N-glycans (5). Glucocerebrosidase, (GCD) is a "memberanebound-lysosomal'' enzyme that stimulates the analysis of glucocerebroside, (GlcCer) into "glucose and ceramide" (6). Gaucher disease is caused by the point mutations in the human GBA gene (Hu-GBA), which results in the aggregation of GlcCer in lysosomes of macrophages (7). The Hu-GBA, first reported in 1985 (8), consists of 497-amino-acids, which came from a 536-mer pro peptide. The mature human glucocerebrosidase (hGCD) has five "N-glycosylation consensus sequences (Asn-X-Ser/Thr)". Glycosylation at the first site is requisite to produce the active protein (9). The native GCD is a glycoprotein containing four carbohydrates chains that does not aims phagocytic cells in the body and therefore has restricted therapeutic value (10). During the development of the present treatment for gaucher disease, the terminal sugars are sequentially removed from the carbohydrate chain of GCD (11) Three different glycosidases involved the formation of a glycoprotein with terminal mannose residues in order to target macrophages which bear mannose receptors (12).While many plant systems have been utilized for backing the expression of heterogeneous proteins, we thought that soybeans may be the most effective of these systems (13). Soybeans are often overlooked as a system of expression in part due to a difficult, lengthy and costly conversion process. However, there is enormous potential for using genetically modified soybeans as a plant to produce pharmaceutical proteins (14). The soybean system has distinctive features that make it a practical alternative to existing expression systems. First, although soybean seeds are traditionally considered highly oiled and proteinaceous seeds, protein represents 38% of the dry mass of soybeans, and is considered one of the richest known natural sources of protein. Offered this high protein content (15), transgenic proteins can be expressed at levels exceeding one milligram in a single soybean seed. There are few, plant systems that are capable of producing such high levels of foreign protein (16). Second, soybeans can be an easy to grow and relatively inexpensive plant. Therefore, the production of biopharmaceuticals in soybeans is very cost effective (17). The aim of this study is to confirm the success of the designed primers with plant signal peptide in amplified the GBA gene and the expression of this gene in soybean plant.

Construction of expression cassette
The pGEM-GBA plasmid from Sino Biological Inc Cat.no.HG12038-G was used as the source for cDNA encoding human glucocerebrosidase (hGCD) (RefSeq: NM_000157), the pCAMBIA 1304 plant expression vector from Marker Gene Technologies, Inc., is controlled by the 35S promoter from cauliflower mosaic virus and terminated by the CaMV35S polyA signal. A genespecific primer pair was designed according to the predicted sequence of GBA (NM_000157; NCBI Reference Sequence). GBA cDNA was then modified a designed forward primer containing DNA coding sequences for the ER targeting signal encoded by the basic endochitinase gene (Arabidopsis thaliana), ATGAAGACTAATCTTTTTCTCTTTCTCATCTT TTCACTTCTCCTATCATTATCCTCGGCCGAA TTC which has been communicated to increase the accumulation of recombinant protein in plant tissues, and a reverse primer containing the vacuolar targeting signal GATCTTTTAGTCGATACTATG from tobacco chitinase A. Recognition sites for the BglII and BstEII restriction enzymes were inserted into the 5′and 3′ends of the designed primers , with the respective recognition site sequences shown in bold as following: The forward primer 5′CTAGATCTATGAAGACTAATCTTTTTCTCT TTCTCATCTTTTCACTTCTCCTATCATTATCC TCGGCCGAATTCGCCCGCCCCTGCA 3′, and reverse primer 5′AGCGGTCACCGATCTTTTAGTCGATACTA TGCTGGCGATGCCACAG3′.

Binary vector construction
The purified PCR product was sequenced and inserted into the binary vector pCAMBIA1304, after being digested with the endonucleases BglII and BstEII, yielding a pCAMBIA1304-GBA vector (Fig. 1). The ligation yield mixture was used to transform the E. coli strain DH5-α (Invitrogen) and kanamycin resistant colonies were segregated after overnight incubation at 37 °C. After amplification using the colony PCR method, the constructed plasmid was extracted from bacterial cells using AccuPrep ® plasmid Mini Extraction Kit (Bioneer), and the plasmid confirmed by restriction enzyme digestion, PCR and sequencing. The plasmid was introduced into A. tumefaciens strain LBA4404 (Takara Bio Inc. Cat. # 9115) by the heat shock method. Transformed cells were screened for kanamycin-resistance and amplified by colony PCR.

Plant materials and cultivation conditions
The Agrobacterium mediated transformation through mature Soybean [Glycine max L.] cotyledon was performed as previously described by (18) and (19) with some modifications.

Sterilization of soybean seeds and germination
Soybean [Glycine max L.] (Seeds were obtained from market in Egypt). Seed surfaces were sterilized using chlorine gas formed by mixing 3.5 mL 12 N HCl with 100 mL bleach (5.25% sodium hypochlorite) for 12 hrs. Sterilized seeds were germinated on basal MS germination medium supplement with B5, 2% sucrose and 0.7% agar (pH 5.8) The tissue culture jars were kept in an incubator at 25°C under 18/6 hrs. (light/dark) photoperiod conditions for 5-6 days.

Preparation of the Agrobacterium strain for infection
A single colony of Agrobacterium containing the pCAMBIA1304-GBA plasmid was cultured for 48 hrs. In yeast extract peptone (YEP) medium supplemented with 100 mg/L streptomycin and 50 mg/L kanamycin. After cells density reaching of OD 600 =1.5, the culture was centrifuged at 5.000 r min−1 for 10 min , and the pellet was resuspended in infection medium (IM) ( 4.33 g /L MS medium supplemented with B5, 30 g/L sucrose

Preparation of explants and infection
When the cotyledons became green and the seed coat split open, they were separated and dipped into the previously prepared Agrobacterium suspension, and shaken at 50 rpm at 28 °C for 30 min. Then, the explants were blotted on sterile filter paper and placed on filter paper laid over the cocultivation medium (CCM), CCM was composed of IM medium supplement with 0.6% agar Cocultivation plates were incubated at 22 °C for 5 days, at 16 hrs. light/8 hrs. dark.

Selection, rooting and hardening
After co-cultivation, Agrobacterium was removed by washing the cotyledons three times with sterile water added with 50 mg/ L carbenicillin, on a shaker, at 410 rpm for 40 min (20). Explants were transferred to selective shoot induction medium (SSIM) composed of full strength MS medium supplemented with 30 g/L sucrose, 3 mM MES,1.68 mg/L BAP, 15 mg/L hygromycin, and 0.65% agar, (pH 5.4) (19) . After 2 weeks, the explants were moved to shoot elongation medium (SEM) full strength MS medium, supplemented with 30 g/L sucrose, 0.750 mg/L gibberellic acid, 3mM MES, glufosinate 5mg/L, 15 mg/L hygromycin and 0.65% agar, (pH 5.8) and cultured under18/6hrs. Light/ dark at 25℃. The explants were sub cultured on the same fresh medium at 14 days intervals. After the formed shoots reached approximately 2-3 cm in height, the regenerating shoots were planted in rooting induction media (RIM) half strength B5 medium was supplemented with 1% sucrose, 2 mg/L indole-3-butyric acid (IBA) and 0.7% agar, (pH 5.4) for 20-30 days. Plantlets were cultivated in rooting media until they developed roots.
Good rooted transformed soybean plants were hardening under controlled environment conditions by wrapping the pots with transparent bags for another weeks under18/6h light/dark at 25°C. The bags were then opened to acclimate for one week. The plantlets were irrigated once in 2days after which they were transferred to soil and vermiculite (1:1) and then grown in a greenhouse under the same climatic conditions. Isolation of Hu-GBA To identify Hu-GBA gene in transformed soybeans total RNA was reverse-transcribed into singlestranded cDNA, using the AccuPower ® RocketScript ™ RT Premix (Bioneer, Korea). Using this cDNA as a template, specific primers (F5-CCATGGCTGGCTGGCATCACA-3 &R5 -CTGGCGATGCCACAG-3) were used to amplify Hu-GBA. The PCR reaction conditions were as follows: 94°C for 5 min, then 30 cycles at 94°C for 1 min, 58°C for 52 sec, and 68°C for 3 min, with a final extension at 68°C for 7 min. The sequences were aligned with the GBA sequence using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

Q R T-PCR assay
Qualitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyse Hu-GBA expression at the transcription level. The RNA was extracted from transformed plant leaf tissue using the GENEzol ™ TriRNA Pure Kit from Geneaid and oligo (dT) primer (Bioneer) to synthesise complementary DNA (cDNA) via reverse transcription, using the AccuPower ® RocketScript ™ RT Premix (Bioneer). The resulting cDNA mixture was used as a template for qRT-PCR. The expression of recombinant GBA was quantitative analysis using a qRT-PCR system (Bioneer). QRT-PCR was performed in a 20 µL reaction volume containing 1 μM of each primer and 10 μL AccuPower ® Green Star ™ qPCR PreMix Bioneer kit). The qRT-PCR experiment was performed using the forward primers 5-CAGCCTCACAGGTTTGCTTCT-3 and reverse primer was R-5GACACACACCGAGCTGTA-3, respectively. The expression of Hu-GBA in transformed plant was compared with the expression of Hu-GBA in the blood of healthy human as a positive control and with gene of untransformed soybean as negative control. The qRT-PCR instrument was programmed as follows: 1 min at 95 °C, 40 cycles of 35 sec at 95°C, 45 sec at 58°C and 1 min. at 68°C, followed by a final extension at 68 °C for 5 min.

Results
The original objective of this study was to modify the sequence of the natural human cDNA encoding for hGCD to permit its expression in soybean. The GCD signal peptide was substituted with that for the Arabidopsis thaliana basic endochitinase gene, and the storage vacuole targeting signal from tobacco chitinase A. The PCR products were analyzed using 1% agarose gel electrophoresis, and specific bands were observed at 1587bp, which included the 1,502 bp of an open reading frame of Hu-GBA, 64 bp targeting signal from basic endochitinase and 21 bp vacuolar targeting signal (Fig.2).

Digestion of the recombinant plasmid with BgIII and BstEII restriction enzymes
After isolated pCAMBIA 1304-GBA, from transformed E. coli DH5-α was double digested with both enzymes BgIII and BstEII, resulted two separated bands (Fig. 3). The top band (9,826 bp) represented the DNA vector without the GUS gene and the bottom band represented the modified GBA with the ER and vacuolar targeting signals (approximately 1,587bp). The recombinant plasmid ''pCAMBIA1304-GBA'' was successfully converted into A. tumefaciens strain LBA4044, used calcium chloride transformed method and the transformed A. tumefaciens LBA4044 was selected on yeast extract peptone YEP medium with kanamycin and streptomycin antibiotics (Fig. 4). The colony PCR was used to scan GBA inserts in transformed bacteria. A 1,587bp fragment of was obtained using specific primers for GBA gene (Fig.5).

Agrobacterium-mediated transformation
During the plant transformations, soybean, half-cotyledons were co-cultivated with Agrobacterium strain harboring the recombinant pCAMBIA1304-GBA plasmid for five days (Fig.  6C). Explants were placed onto the SIM for two weeks to promote the shoot formation (Fig. 6D). Two weeks later, shoots were transferred to SEM (Fig. 6E). After four to six weeks, the elongated shoots were transferred to RIM (Fig. 6F). Rooted plantlets were transplanted into small pots, containing soil mixture initially were covered with plastic dome and placed in an incubated (Fig.6g, h). The resulting T0 plantlets were then transplanted into bigger pots and grown in soil in a greenhouse (Fig. 6i). In this study, the transformation of Hu-GBA in soybean was verified. Glycosylated proteins can be produced using a plant culture, particularly proteins with high levels of mannose glycosylation, and the proteins can be designed to target the ER and/or by-pass the Golgi using specific vectors resulting in the production of enzymatically active, high-mannose lysosomal enzymes using transgenic plants. In this study the nucleotide sequence of the Hu-GBA gene was modified, through PCR techniques, using designed primers, to fuse the signal peptide from Arabidopsis thaliana basic endochitinase and a C terminal vacuole targeting sequence from tobacco chitinaseA, to the Hu-GBA the sequence, as described by a previous study (20). Targeting the heterogeneous proteins to the appropriate organelles can be crucial to obtaining high levels of accumulation because the structure and stability of a recombinant protein are determined by its pathways and endpoints in the cell. The ER is the entry port for the secretory protein pathway (21), and the ER is the site where multiple newly synthesized peptides multiply and assemble and glycoproteins become glycosylated on aspartame residues (22). A 1,587bplong PCR product was cloned upstream from CaMV35S and downstream of the NOS terminator in the pCAMBIA1304 vector between BstEII and BgIII restriction sites (Fig.1). This construct was transformed into the A. tumefaciens strain LBA4404 after being isolated from transformed E. coli DH5α cells. The presence of Hu-GBA was confirmed by colony PCR (Fig. 3) and digestion reactions, which were conducted using the BstEII and BglII restriction enzymes (Fig.4). Agrobacterium infection plays a major role in the soybean transformation processes (23). Researchers have demonstrated that the cotyledonary node is good candidate explant for shoot regeneration (24). In Agrobacterium-mediated transformation systems, the cotyledonary node must first be wounded to release phenolic compounds and to provide access to the target cell. The wound enhances the activity of the internal cytokines to stimulate cell division (18). In this study, the cotyledons were cut because wounding is important for increasing the efficient plant transformation, as Agrobacterium can sense a wounded potential host by perceiving these phenolic compounds. The infection medium also contained AS and DTT and had an acidic pH, which are all factors that have been shown to facilitate T-DNA transfer and to enhance transformation efficiency (25). After 5 days of co-cultivation the moderate survival of explants was observed but with higher transformation efficiencies (26). In this study, DTT was added to the CCM as an antioxidant, to reduce pruning and necrosis in explants which represents one of the primary factors affecting the conversion efficiency (19). Cytokinins can activate cell division, and many reports have shown a direct link between cytokinins and cell cycle control (27). The transgenic soybeans were analysed by PCR and qRT-PCR, and these techniques demonstrated the steady expression of recombinant GBA in transgenic plants, at the RNA level. GBA could be amplified from genomic RNA isolated from transformed soybean leaves, using specific primers, as shown in (Fig.7), appeared to have the same band size as the control GBA which proved that the gene that amplified from transgenic plants was GBA. The q RT-PCR results (Fig. 8) showed that GBA was expressed in transformed soybeans plants, at relatively lower levels than in human blood. No GBA RNA was detected in nontransformed control plants. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the preferred method for confirming gene expression and detecting the fold change difference in mRNA levels for any target gene, due to its high sensitivity, specificity and reproducibility (28). The expression of GBA gene in transformed soybeans was confirmed when its RNA we converted to cDNA, and the GBA expression level was compared with than of blood sample from a healthy person. GBA mRNA in transgenic soybean plant was closed to its level in the human blood, and this may be an indication of the possibility of producing the glucocerebrosidase from soybeans and considering it as economic source for this enzyme.

Conclusions:
In this study, Agrobacterium successfully inserted Hu-GBA inside soybean genomic and generated transformed plants. In this protocol, the pCAMBIA 1304 plant expression vector was used as binary vector, soybean cotyledons were selected as explants. CDNA-GBA was modified using designed primers that contained both an ER targeting signal, and a vacuolar targeting signal. The qRT-PCR analysis showed that transgenic soybeans effectively expressed Hu-GBA with comparable levels as compared with those observed in healthy human blood.