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.