Bioproducción de igf-1

Resumen

BIOPRODUCCION DE IGF-I RESUMEN: Bio production OF iGF-I insu1in growth factor-i (igf-i) is a hormone with potential pharmaceutical application, it is a protein mainly produced by the liver that mediates most of the physiological actions of the growth hormone. investigators at the University of Navarra has demonstrated previously that substitutive treatment with igf-i in liver conditions, such as experimental cirrhosis in rodents, improves nutritional status and prevents the atrophia seen in various tissues in these illnesses. The bioactivity of igf-I requires large quantities of this protein for long term treatment of patients. New biotechnological systems to optimize production are needed in this work the gene encoding mature/active igf-i cdna has been inserted into two bioproduction systems and two new techniques of affinity chromatoqraphy have been implemented. The two systems explored were transient infection of cell cultures with recombinant Semliki forest virus and tobáceo plants made transgenic in their chloroplast dna (Plastidial transformation). The following conclusions have been reached: l. Semliki forest virus (sfv) based vectors are capable of expressing the mature form of human igf-I or fusion proteins of igf-I with the ZZ domain from S.Aureus protein A 2. The sfv-igf-1 vector was capable of expressing up to l g of igf-I active protein per million of BHK cells tat was collected in the tissue culture médium. 3. The sfv vectors encoding ZZTEV-igf-1 and ZZ-ha-igf-i expressed up to 5.2 mq of recombinant protein per million of BHK cells that was active in functional assays 4. The conditions for the affinity chromatography have been optimized in their pH, time course and temperature for digestions and elution. Such conditions were optimized in Ig-coated sepharose columns both for fusion proteins 5. The yield of purification of igf-I was in the best of the cases of 25% using zz-ha-igf-i as the raw material. Moreover, less than 1% of such igf-i was active for signalling through stats and AKT 6. Two tobáceo transgenic plants have been produced Through the technique of chloroplast DNA transformation. One of them expresses ZZ-tev-igf-I while the other expresses ZZ-HA-igf-i. The levies of expression of these plants were 1-12 g/gr of fresh plant. These values were considered too low to use them as a source for purification