A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

Abstract

We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.A host-vector system for the efficient extracellular production of heterologous proteins, involving Bacillus brevis as the host, has been developed (22). Many proteins of bacterial origin could be produced at high levels without much difficulty. However, certain proteins, especially some mammalian ones, still exhibited low productivity. Sagiya et al. improved the B. brevis protein expression system by means such as modification of the signal sequence (17) and isolation of a protease-deficient mutant (7), which was successfully applied to the secretion and accumulation of not only prokaryotic but also eukaryotic proteins. In fact, a change of the signal peptide sequence in the B. brevis system resulted in the higher secretion of many proteins, for example, growth hormones (7, 17), interleukin 2 (21), and protein disulfide isomerase (PDI) (8), and the use of proteasedeficient mutants increased the production of extracellular proteins (6, 7). Still another way of increasing protein productivity is to link the gene of interest to a second gene which is already known to be expressed well in the host to generate a fusion protein (9). In most of the successful fusion protein systems the protein of interest is positioned at the C-terminal end of the highly expressed fusion partner (12, 19) to ensure efficient translation initiation. The thioredoxin gene fusion system also provided the solution for another major problem which has bedeviled heterologous gene expression in E. coli, i.e., the formation of inclusion bodies (11). PDI catalyzes the formation, reduction, and isomerization of disulfide bonds in vitro (5) and facilitates the folding of disulfide-bonded proteins in vivo (18). Even though the physiological roles of these multiple functions of PDI during protein folding in the cell remain obscure, PDI, promoting in vitro folding, can be used to improve an expression system for foreign genes. In fact, in a coexpression system, PDI exhibits chaperone-like activity which suppresses the aggregation and increases the yields of heterologou...

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