Agrobacterium-mediated transformation of lettuce (Lactuca sativa L.) to express IgG-binding protein A and human pro-insulin as a fusion protein.

The worldwide demand for pro-insulin increases continuously as new medical applications are developed. Lettuce (Lactuca sativa L.) has the potential to help supply this rising demand as a "green bio-reactor". A DNA construct containing the immunoglobulin G-binding protein A gene of Staphylococcus aureus (ProA) genetically fused to a nucleotide sequence encoding pro-insulin (Pins) was produced and transformed into lettuce. Integration of the ProA-Pins fusion gene into the chromosomes of transgenic lettuce, and copy numbers were verified by PCR amplification of genomic DNA and Southern blotting, respectively. Expression of the ProA-Pins fusion protein gene in transgenic lettuce was confirmed by reverse transcription-PCR analysis. Western immunobloting analysis of the proteins extracted from the leaves of transgenic lettuce plants revealed the presence of an immuno-reactive band with a molecular weight of approx. 20 kDa using rabbit polyclonal antibody against insulin. The plant-produced ProA-Pins fusion protein gave significant readings for indirect enzyme-linked immunosorbent assays (ELISA) using rabbit polyclonal antibody against insulin. The highest level of ProA-Pins expression in transgenic lettuce plants was 0.13% (w/w) of total soluble protein. This work demonstrated that plant-based systems expressing pro-insulin may provide an effective, low-cost approach to produce insulin for the treatment of Types I and II diabetes. [ABSTRACT FROM AUTHOR]