Efficient production of recombinant human serum albumin in Escherichia coli via oxidative folding mediated by the a domain of protein disulfide isomerase.

Efficient production of properly folded, disulfide-rich proteins in the cytosol of Escherichia coli remains challenging because the cytosol is highly reducing. Here, we developed an expression system for recombinant human serum albumin (rHSA) by fusing the a domain of human protein disulfide isomerase (PDI) to the N-terminus of HSA. PDI(a)-mediated cis-fusion enabled high-level production of soluble rHSA in oxidizing E. coli strains under low-temperature conditions. Purified E. coli-rHSA was predominantly monomeric and had the molecular mass expected for HSA containing 17 intramolecular disulfide bonds. E. coli-rHSA retained esterase-like activity comparable to that of rHSA produced in Pichia pastoris (P. pastoris-rHSA) and serum-derived HSA, confirming preservation of this activity. Circular dichroism analyses and small-angle X-ray scattering showed that E. coli-rHSA adopted a solution structure nearly identical to that of P. pastoris-rHSA. Disulfide-bond mapping identified 13 of the 17 native disulfide bonds, although alternative pairings were also detected. E. coli-rHSA showed slightly lower thermal stability than P. pastoris-rHSA under nonreducing conditions. These results demonstrate that PDI(a)-mediated cis-fusion enables high-yield production of structurally native-like and functionally competent HSA in the bacterial cytosol and provides a practical platform for producing complex disulfide-rich proteins in E. coli.