Human insulin-like growth factor I (TGF-I) accumulates in both folded
and aggregated forms in the fermentation medium and cellular periplasm
ic space when expressed in E. coil with an endogenous secretory signal
sequence. Due to its heterogeneity in form and location, low yield of
IGF-I was obtained using a typical refractile body recovery strategy.
To enhance recovery yield, a new procedure was developed to solubiliz
e and extract IGF-I from cells while in fermentation broth. This metho
d, called in situ solubilization, involves addition of chaotrope and r
eductant to alkaline fermentation broth and provides recovery of about
90% of all IGF-I in an isolated supernatant. To further enhance recov
ery, a new aqueous two-phase extraction procedure was developed which
partitions soluble non-native IGF-I and biomass solids into separate l
iquid phases. This two-phase extraction procedure involves addition of
polymer and salt to the solubilization mixture and provides about 90%
recovery of solubilized IGF-I in the light phase. The performance of
the solubilization and aqueous extraction procedures is reproducible a
t scales ranging from 10 to 1000 liters and provides a 70% cumulative
recovery yield of IGF-I in the isolated light phase. The procedure pro
vides significant initial IGF-I purification since most host proteins
remain cell associated during solubilization and are enriched in heavy
phase. ELISA analysis for E. coli proteins indicates that 97% of the
protein in the light phase is IGF-I. Together, the techniques of in si
tu solubilization and aqueous two-phase extraction provide a new, high
yield approach for isolating recombinant protein which is accumulated
in more than one form during fermentation.