Streptomyces griseus protease B: secretion correlates with the length of the propeptide

Abstract

Streptomyces griseus protease B, a member of the chymotrypsin superfamily, is encoded by a gene that express a pre-pro-mature protein. During secretion the precursor protein is processed into a mature, fully folded protease. In this study, we constructed a family of genes which encode deletions at the amino-terminal end of the propeptide. The secretion of active protease B was seen to decrease in an exponential manner according to the length of the deletion. The results underscore the intimate relationship between folding and secretion in bacterial protease expression. They further suggest that the propeptide segment of the zymogen stabilizes the folding of the mature through many small binding interactions over the entire surface of the peptide rather than through a few specific contacts.

FIG. 1

Relative production of SGPB from mutant sprB genes. Expression constructs encoding SGPB with different numbers of amino acids (shown following Δ) deleted from the N terminus of the propeptide were used. Protease activities (░⃞) and immunodetectable protein (▪) are shown relative to those of the wild type (encoded by pEB-B8). The vector (encoded by pEB-11) contains no sprB insert. The chimera (encoded by pEB-PAmB) is the proA-mature B fusion.

FIG. 2

Effect of temperature on mutant SGPB activity. B. subtilis cultures harboring pEB-BPΔ10, pEB-BPΔ15, pEB-BPΔ20, or pEB-11 were grown for 59 h at 30°C. Activity is plotted as a log function of the number of amino acids deleted from the propeptide for cultures grown at 21 (•), 30 (○), and 37°C (×).

FIG. 3

Identification of extracellular SGPB. A Western blot probed with anti-SGPB is shown. B. subtilis expression constructs encoding different numbers of amino acids (shown following Δ) deleted from the N terminus of the propeptide of SGPB were used. B, purified SGPB. The chimera (encoded by pEB-PAmB) is the proA-mature B fusion. The wild type is encoded by pEB-B8. The vector (pEB-11) contains no sprB insert.

FIG. 4

Recombinant expression by chimeric proA-mature B protease. B. subtilis DB104 transformants harboring pEB-pAmB (chimera) (1), pEB-B8 (wild type) (2), or pEB-11 (vector control) (3) were grown on YT-kanamycin agar supplemented with 1.5% skim milk and incubated for 18 h at 30°C. The diameter of each zone of clearing is proportional to the level of proteolytic activity.

FIG. 5

Helical wheel representations of amino acids 7 to 24 of the propeptide of SGPA (A) and the homologous amino acids 5 to 22 of the propeptide of SGPB (B) (8). The analysis was generated with the computer program MacDNASIS Pro (Hitachi), with 3.6 amino acids per turn. Dark shading, chemically identical amino acids; light shading, chemically similar amino acids.