Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide

Abstract

Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to ~40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyper-expression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.

Figure 1

Location of furin cleavage sites. N and C termini are indicated. (a) Native proinsulin. (9 kDa) (b) Proinsulin molecule was modified to include furin cleavage sites and fused to cholera toxin B subunit (CTB). MW of CTB is 11 kDa. MW of modified CTB-proinsulin molecule is 22 kDa. Fusion proteins are expressed by the CTB-Fx3Pris coding region of the pLDutr-CTB-Fx3Pris or pLsLF-CTB-Fx3Pris chloroplast vector.

Figure 2

Southern analysis of transgenic tobacco containing cholera toxin B subunit (CTB)-Proinsulin with three furin cleavage sites. (a) Map of untransformed tobacco plastome. Digestion with AflIII yields a 4.2-kb fragment. Broken line represents probe annealing site. (b) Map of transformed tobacco plastome. Digestion with AflIII yields a 6.4-kb fragment. The 5′UTR represents the light-regulated psbA promoter. (c) Southern analysis from T₁ generation. A and B represent independent transplastomic lines. WT, untransformed plant.

Figure 3

Southern analysis of transgenic lettuce containing cholera toxin B subunit (CTB)-proinsulin with three furin cleavage sites. (a) Map of untransformed lettuce plastome. Digestion with BglII yields a 3.75-kb fragment. Broken line represents probe annealing site. (b) Map of transformed lettuce plastome. Digestion with BglII yields a 6.3-kb fragment. (c) Southern analysis from T₀ generation. 1, 2, 5, 6 and 8 represent independent transplastomic lines. Lowercase letters a and b represent different samples from the same line. WT, untransformed plant.

Figure 4

Characterization of cholera toxin B subunit (CTB)-proinsulin expressed in tobacco chloroplasts. (a) Coomassie stain. M, marker; S, supernatant; P, pellet; H, homogenate; WT, untransformed plant. S and H: 30 μg per lane. P: 7.5 μg per lane. (b) Western blot probed with anti-CTB antibody showing CTB-PFx3 expression of young, mature and old leaf total protein. 1 μg per lane. CTB: 100 ng CTB standard. Arrows represent the monomer, dimer and trimer bands, respectively. (c) CTB-PFx3 total protein was loaded in varying concentrations and compared to known quantities of CTB standard protein using densitometry. Blot was probed using anti-CTB antibody. (d) Plot of integrated density values (IDV) for quantification of CTBPFx3 based on standard curve. Broken line shows data points. Solid line: trend line. (e) Per cent total protein for young, mature and old leaves based on densitometry values. (f) Quantification of young, mature and old leaves in mg CTB-PFx3 per gram total leaf tissue.

Figure 5

Characterization of cholera toxin B subunit (CTB)-proinsulin expressed in lettuce chloroplasts. (a) CTB-PFx3 total protein was loaded in varying concentrations and compared to known quantities of CTB standard protein using densitometry. Blot was probed using anti-CTB antibody. (b) Plot of integrated density values (IDV) for quantification of CTB-PFx3 based on standard curve. Broken line shows data points. Solid line: trend line. (c) Per cent total protein for young, mature and old leaves based on densitometry values. (d) Quantification of young, mature and old leaves in mg CTBPFx3 per gram total leaf tissue.

Figure 6

Expression of cholera toxin B subunit (CTB)-proinsulin in dried senescent lettuce leaves. (a) CTB-PFx3 total protein from dried leaf homogenate was loaded in 1 or 2 μg concentrations along with varying dilutions of CTB standard protein. Immunoblot was probed with anti-CTB antibody. (b) CTB-PFx3 total protein from dried leaf homogenate was loaded in 1 or 2 μg concentrations along with varying dilutions of insulin standard protein (shown as multimers). Immunoblot was probed with anti-insulin antibody.

Figure 7

Furin cleavage assays. Samples were solubilized with 6 M Gu-HCl and 300 mM DTT and subsequently dialysed in 20 mM Na⁺ PO₄⁻ buffer with 500 mM NaCl before addition of Furin protease. (a) Furin digestion of CTB-PFx3. CTB, 100 ng CTB standard protein. −F, CTB-PFx3 before furin cleavage. +F, CTB-PFx3 after addition of furin. 1 μg per lane. (b) Silver stained gel after affinity purification of CTB-PFx3. Lane 1, precision plus ladder. Lane 2, Mark 12 low molecular weight ladder. Lane 3, purified CTB-PFx3 1 μg per lane. Lane 4, purified CTB-PFx3 after addition of furin. 1 μg per lane.

Figure 8

Purification of cholera toxin B subunit (CTB)-proinsulin by affinity chromatography. (a) Coomassie stained gel. DS = dialysed supernatant; FT = flow through; W1 = wash 1; W2 = wash 2; 2,3 = purified fractions; WT = untransformed plant material; CS = control supernatant; CP = control pellet (b) Coomassie stained gel showing second purification of CTB-PFx3: Lane 1: Marker. Lane 2: control pellet (before purification). Lane 3: Purified CTB-PFx3, Lane 4: Purified CTB-PFx3 after phytate precipitation to remove Rubisco. Lane 5: Control supernatant. All 10 μg load. (c) 1 μg of purified CTB-PFx3 samples were loaded using CTB primary antibody. DS = dialysed supernatant; FT = flow through; W1 = wash 1; W2 = wash 2; 2, 3 = purified fractions (d) 1 μg of purified CTB-PFx3 samples were loaded using insulin primary antibody. DS = dialysed supernatant; FT = flow through; W1 = wash 1; W2 = wash 2; 2, 3 = purified fractions (e) Densitometry shows that after purification, CTB-PFx3 makes up ~57% of the eluted fractions after the first purification and ~87% after the second purification as compared to ~27% of the dialysed solubilized supernatant before passing over the nickel column. (f) The concentration of CTB-PFx3 increases roughly 10-fold (~320 000 ng/mL) after the first purification and 4.6-fold (~140 000 ng/mL) after the second purification over nickel column than the initial concentration of dialysed solubilized supernatant (~30 000 ng/mL).

Figure 9

Functional evaluation of cholera toxin B subunit (CTB)-proinsulin in mice delivered by injection or oral gavage. ANOVA (Dunnett’s multiple comparison test) was performed on all groups of mice. (a) Mice were administered two intraperitoneal (IP) injections of PBS only (PBS), purified CTB-PFx3 (Tg) or commercial Insulin (INS Comm.). Blood glucose levels were measured after the first 15 min and each hour (up to three hours) after each injection. Blood glucose levels were statistically lower (**P < 0.01) 2 h after the second injection in all groups compared to the PBS only negative control. (b) Mice were administered two oral gavages of untransformed lettuce (WT), tobacco expressing CTB-PFx3 (Tg tobacco), lettuce expressing CTB-PFx3 (Tg lettuce) or two IP injections of commercial Insulin (INS Comm.). Blood glucose levels were measured after the first 15 min and each hour (up to three hours) after each injection. Blood glucose levels were statistically lower (**P < 0.01; ***P < 0.001) 2 h after the second injection in all groups compared to the untransformed (WT) control.