PARENT SESSION

Symposium S7D Chloroplast factories and transformation
Thursday September 2nd, 2004 2:40 PM-4:40 PM Room 510A
Chair: Dick Sayre
Co-Chair: Pal Maliga

High level expression of recombinant proteins through chloroplast genetic engineering and their purification. Sadhu Leelavathi, Amit Bhardwaj, Vijay Kant, Sachin Gupta, Vanga Reddy^*,,

ABSTRACT- During the past one decade the ability of transgenic plants to produce a variety of recombinant proteins has been extensively demonstrated. However, large-scale production of recombinant proteins in plants is currently hampered by relatively low levels of transgene expression and lack of efficient and cost-effective downstream process. During the last couple of years we have been working to overexpress a number of recombinant proteins that are useful in industry and in human health using chloroplast genetic engineering and fusion-protein strategies. Such strategies allowed us to over express human interferon gamma, an important therapeutic molecule in pharmaceutical industry and a cell wall degrading xylanase from Bacillus subtilis, useful in paper and animal feed industry at high levels (8% of total soluble protein). In addition to overexpression, strategies were developed to simplify the downstream process for efficient recovery of recombinant proteins. For this purposes, recombinant protein was engineered to express as a fusion protein of proteolytically cleavable His-tag and purified using affinity (Ni-NTA) based chromatography, essential for cost-effectiveness. Also, purification based on the thermo-stability of the recombinant protein imparted by a thermostable enzyme as a fusion partner was tested. In addition, an inducible expression based on the T7 RNA polymerase depended transcription was developed to express Hepatitis B surface antigen (HBsAg), an edible vaccine candidate, in tobacco chloroplasts. Implication of these results on the large scale production and purification of recombinant proteins using transplastomic plants will be discussed.

KEY WORDS: Recombinant proteins, Chloroplast genetic engineering, Protein purification, Inducible expression