Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin.

When the mammalian aspartic proteinases, procathepsin D or pepsinogen, are expressed in Escherichia coli both accumulate in inclusion bodies. While pepsinogen is efficiently refolded in vitro, recovery of procathepsin D is limited by insolubility. We expressed procathepsin D and pepsinogen in E. coli, with E. coli maltose-binding protein (MBP) or thioredoxin (trx) fused to their C-termini (aspartic proteinase-MBP or aspartic proteinase-trx). The fusion proteins were still found in inclusion bodies. However, the recovery of soluble procathepsin D-MBP and procathepsin D-trx after refolding was facilitated by the bacterial fusion partners. Maltose-binding protein was more efficient than thioredoxin in increasing the recovery of soluble protein. The vector, pET23bMBPH6, can be used for general expression of heterologous proteins in E. coli. The vector includes a histidine tag at the C-terminus of MBP to allow one-step purification of the fusion proteins under denaturing conditions. After purification, the protein of interest can be cleaved from MBP with factor Xa protease and separated from the MBP partner. Refolded pepsinogen-MBP and pepsinogen-trx were enzymatically active, but procathepsin D-MBP and procathepsin D-trx were soluble but largely inactive. The results show that the limited recovery of activity upon refolding of procathepsin D is not the consequence of competing aggregation. Thus, the fusions do not necessarily facilitate native refolding, but they do enhance the recovery of soluble protein. Such fusions could provide a system to study, in soluble form, folding states which are otherwise inaccessible because of aggregation and precipitation. Copyright 1998 Academic Press.