PRODUCTION OF RECOMBINANT HTLV-I TRANSMEMBRANE PROTEIN (RP21E)

Abstract

The culture and induction conditions for the expression of human T-cell lymphotropic virus type-I (HTLV-1) transmembrane protein (p21E) by recombinant E. coli MZl (pKS400), were studied in both shake flask and fermenter cultures. The effects of early and late induction on rp21E yield, productivity and protein quality were investigated. A carbon-source limited Complex Medium (CM) containing basal salts, trace metals and glucose to yeast extract in an optimal ratio determined to be 1:3.3 was developed using central composite and fractional factorial design experiments. This medium was found to be more cost effective than Super Broth (SB), the original medium recommended for the cultivation of this recombinant microorganism. The overall cell yields based on glucose, histidine, valine and isoleucine (g DCM/g substrate) were experimentally determined to be 1.45, 50, 18 and 18 respectively. Batch and fed-batch fermentations using the designed medium resulted in maximum OD of 96 and 66 respectively. In the latter case, higher cell density can be achieved if the feeding is extended before induction. Both shake flask and fermenter studies showed that cultures induced during early log phase resulted in higher rp21E yields than cultures induced later. However, because of higher cell densities, the volumetric productivities were higher in the cultures induced during late log phase. The induction time for maximum rp21E yield was found to be 3 to 4 hours. High cell densities, with concomitantly high yields (51-65 mg rp21E/g DCM) and productivities of rp21E (31- 54 mg rp21E/h/L), were achieved in both batch and fed-batch fermentations. The results obtained from protein sequencing, in situ trypsin digestion and peptide mapping showed that the rp21E produced from batch (early and late induction) and fed-batch cultures was of similar protein quality. Western immunoblotting showed that the rp21E produced retained viral antigenicity.