Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment fromBacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

Abstract

To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts

“…subtilis is able to secrete several proteins up to 20 g l −1 (Schallmey et al, 2004), however it is know that secretion of heterologous proteins in B. subtilis is usually lower (Li et al, 2004). A frequently encountered problem with expression and secretion of heterologous proteins in B. subtilis is the rapid degradation of the secreted protein by extracellular proteases (Wu et al, 2002). It has also been reported that ␤-toxoid is sensitive to protease activity (Hunter et al, 1993).…”

“…It is therefore possible that ␤-toxoid is degraded when in it is secreted into the medium. To test this, the expression cassette was integrated in the amyE locus of a B. subtilis strain WB700 in which seven of the major extracellular proteases are deleted (Wu et al, 2002). In this strain, secreted ␤-toxoid was detected in the medium at concentrations of 0.1-0.5 g ml −1 culture.…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…• Development of expression systems able to drive gene expression under stress conditions • Co-administration of mucosal adjuvants either for biotechnological or biochemical applications (Simonen and Palva 1993, Udaka et al 1989, Udaka and Yamagata 1993, Wang et al 1988, Wong 1995, Wu et al 2002, Wu and Wong 1999. Strong promoters with tightly regulated activity, as those induced by xylose or IPTG, are available for B. subtilis and some of them have already been tested for expression of heterologous or endogenous proteins either as intracellular or secreted proteins (Conrad et al 1996, Kim et al 1996.…”

“…Strong promoters with tightly regulated activity, as those induced by xylose or IPTG, are available for B. subtilis and some of them have already been tested for expression of heterologous or endogenous proteins either as intracellular or secreted proteins (Conrad et al 1996, Kim et al 1996. Similarly, B. subtilis strains unable to secrete several proteases have been shown to reduce degradation of secreted heterologous proteins to trace levels, as compared to the previously used laboratory strains, and can be used as more appropriate hosts for the production of vaccine antigens (Wu et al 1991(Wu et al , 2002.…”

“…In contrast to chromosome integration methods, enhanced structural and segregational gene expression could be achieved with such vectors without reduction of gene copy number (Jannière et al 1990). The identification of secreted and cell-bound proteases of B. subtilis permitted the generation of strains defective in 6, 7 or 8 different proteases, which greatly improved the recovery of recombinant proteins from the growth medium of genetically modified strains (Wu et al 1991(Wu et al , 2002. The complete sequencing of the B. subtilis genome and the availability of promoter trap systems greatly simplified the task of identification and evaluation of promoters with potential applications for heterologous gene expression (Gat et al 2003).…”