Engineering subtilisin BPN′ for site‐specific proteolysis

Abstract

A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support.

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 Å resolution

“…The enzymes within each individual family have diverged from a common ancestor, but the two families show no similarity in sequence or three-dimensional structure and appear to have converged on the same solution for proteinase activity [ 11. The serine proteinases have been extensively studied by a variety of biochemical and genetic techniques and their mechanism and specificity is well understood [2,3]. In addition the three-dimensional structures have been determined for several enzymes from both families, as well as their complexes with synthetic and naturally occurring protein inhibitors and for many site-directed mutants [4-71. In spite of the difference in protein fold for the two families their binding sites show certain similarities.…”

Engineering multiple properties of a protein by combinatorial mutagenesis.

“…Recombinant DNA technology has allowed the construction of proteins of altered stability in vitro (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), catalytic efficiency (17)(18)(19), substrate specificity (20)(21)(22)(23), and resistance to in vivo thermal inactivation (24) through the use of single or multiple amino acid substitutions. This effort has been greatly helped by the fact that the effects of amino acid substitutions on such properties of proteins tend to be additive as mutations accumulate, provided that the substituting residues do not interact functionally or by direct contact (25).…”

Generation of engineered recombinant hepatocyte growth factor cleaved and activated by Genenase I

“…This was coupled with a conversion from biologically inactive to active HGF. Since recombinant Genenase I can be produced as an active enzyme using bacteria such as Bacillus subtilis (Carter et al, 1989), our finding provides a novel technique for the preparation of biologically active, two-chain HGF completely under serum-free conditions. One potential concern with medical application of the present technique is the antigenicity of engineered HGF due to the substitution of amino acids at the junction site.…”

“…To achieve this, we designed engineered human pro-HGFs that contain specific amino acid sequences at the cleavage site between the α-and β-chains of HGF so that single-chain pro-HGFs can be cleaved by a specific protease. We selected Genenase I (Carter et al, 1989;Carter et al, 1991), a H64A mutant of subtilisin, as the specific protease for four reasons: 1) the minimum amino acid sequence recognized by Genenase I is only His-Tyr or Tyr-His; 2) neither His-Tyr nor Tyr-His are found in the human HGF sequence; 3) Genenase I can be produced in an active form without serum; and 4) the enzyme reaction can be achieved under mild conditions (e.g., does not require reducing conditions, which inactivate HGF).…”