This study investigates the expression and separation of a recombinant HIV/-beta-gal fusion protein expressed in Escherichia coli under trp promoter control. Product expression and its sensitivity to proteolytic degradation are correlated with inducer strength and protease activity. Two previously unreported proteolytic activities of 42 and 55 kDa molecular weight were revealed which were shown to have activity towards beta-gal. Furthermore, by taking advantage of the extreme partitioning behavior of beta-galactosidase, the HIV-beta-gal fusion was separated and partially purified in a polyethylene glycol-potassium phosphate aqueous two-phase system. Additionally, the 55 kDa protease with beta-gal specificity partitioned into the salt-rich bottom phase.