Plant Expression of Human Proinsulin Using the Plasmid Viral University of Tehran (pVUT) Vector

Insulin is a hormone exclusively produced by pancreatic beta cells. The production of recombinant proteins in microorganisms has some disadvantages such as high cost, the possibility of contamination with toxic proteins, and costly purification steps; so, the production of recombinant proteins in plants can be investigated. Development of transient expression system based on a deleted version of Cowpea mosaic virus RNA-2, Cowpea Mosaic Virus-Hyper Translatable (CPMV-HT), has provided the extremely high-level and rapid production of proteins without viral replication. In this study, two constructions were prepared; pBI121-Proinsulin-Zera (pBI-ProZ) containing human proinsulin gene and Zera (N-terminal proline-rich domain of γ-zein) and pCAMBIA1304-Proinsulin-Extensin (pCAMBIA-ProE) containing CPMV-HT expression system for improvement of translation of human proinsulin gene and carrot extensin signal peptide. Both structures were transiently transferred in to lettuce and alfalfa leaves using Agrobacterium tumefasiens pv. C58. Statistical analysis of this study was conducted on the concentration of produced proinsulin per each gram of transgenic leaf using factorial arrangement of treatment in a complete randomized design. Gene expression was confirmed in transcription level using reverse transcription polymerase chain reaction (RT-PCR) method and in translation level using enzyme-linked immunosorbent assay (ELISA) and Dot blot assay. Protein accumulation for pCAMBIA-ProE and pBI-ProZ constructs were 6.82 and 4.32 ng/g in recombinant alfalfa leaves and 6.6 and 3.8 ng/g in recombinant lettuce leaves, respectively. Results showed that the expression of proinsulin in pCAMBIA-ProE contains CPMV-HT expression system was more than pBI-ProZ.