Staphylokinase as a plasminogen activator component in recombinant fusion proteins

Abstract

The plasminogen activator staphylokinase (SAK) is a promising thrombolytic agent for treatment of myocardial infarction. It can specifically stimulate the thrombolysis of both erythrocyte-rich and platelet-rich clots. However, SAK lacks fibrin-binding and thrombin inhibitor activities, two functions which would supplement and potentially improve its thrombolytic potency. Creating a recombinant fusion protein is one approach for combining protein domains with complementary functions. To evaluate SAK for use in a translational fusion protein, both N- and C-terminal fusions to SAK were constructed by using hirudin as a fusion partner. Recombinant fusion proteins were secreted from Bacillus subtilis and purified from culture supernatants. The rate of plasminogen activation by SAK was not altered by the presence of an additional N- or C-terminal protein sequence. However, cleavage at N-terminal lysines within SAK rendered the N-terminal fusion unstable in the presence of plasmin. The results of site-directed mutagenesis of lysine 10 and lysine 11 in SAK suggested that a plasmin-resistant variant cannot be created without interfering with the plasmin processing necessary for activation of SAK. Although putative plasmin cleavage sites are located at the C-terminal end of SAK at lysine 135 and lysine 136, these sites were resistant to plasmin cleavage in vitro. Therefore, C-terminal fusions represent stable configurations for developing improved thrombolytic agents based on SAK as the plasminogen activator component.

FIG. 1

(A) Amino acid substitutions in the SAK variants constructed. Only the amino acids that are different from the amino acids in the SAK 42D (13) sequence in panel B are indicated; the remaining amino acids are the same as the amino acids in SAK 42D. The region deleted from ΔN₁₀SAK is indicated by a line. (B) Components of expression-secretion plasmid pSAKP. The amino acids boardering the SacB signal sequence and SAK junction are indicated, as are the primer sites used for PCR amplification of the different SAK site-directed variants. Lysine residues described in the text are numbered. The signal peptidase cleavage site is indicated by the open arrow. The primary plasmin cleavage site in SAK is indicated by the solid arrow.

FIG. 2

Flowcharts for construction of the HV1-SAK (A) and SAK-HV1 (B) expression vectors. The restriction enzyme sites used in construction (see text) are indicated. P43, P43 promoter; SacB SP, levasucrase signal peptide, including a ribosomal binding-translation initiation site; linker, synthetic linker peptide.

FIG. 3

(A) Amino acids at boundaries and junctions for each protein domain in HV1-SAK. The HV1 (13) and SAK 42D (1) protein sequences have been published previously. The arrow indicates the plasmin processing site between Lys-10 and Lys-11 in SAK. (B) Coomassie blue-stained protein gel containing HV1-SAK samples which were processed by plasmin at various reaction times (0 to 32 min) and comparative protein samples, including plasminogen (Plgn), HV1-SAK, ΔN₁₀SAK, and SAK. Samples were prepared in a reducing buffer. (C) Anti-SAK Western blot detection of processed HV1-SAK, ΔN₁₀SAK, and SAK.

FIG. 4

Coomassie blue-stained protein gels showing plasmin processing of SAK and SAK variants after reaction for 0 to 20 min. Lanes contained the following protein samples: plasminogen (lane Plgn), the SAK variant (lane SAK∗), and ΔN₁₀SAK (lane ΔN₁₀SAK). Processing of the wild-type SAK is shown in the top gel. The SAK variants used in this study are indicated beside the corresponding gels.

FIG. 5

(A) Amino acids at the boundaries and junctions for the protein domains in SAK-HV1. The solid arrow indicates the primary plasmin processing site between Lys-10 and Lys-11 in SAK. The open arrows indicate the Lys-135 and Lys-136 residues that were tested for plasmin sensitivity. (B and C) Coomassie blue-stained protein gels containing HV1-SAK processed by plasmin for 0 to 32 min. The proteins used for comparison were plasminogen (Plgn), HV1-SAK, ΔN₁₀SAK, and SAK. Samples were prepared under nonreducing (B) and reducing (C) buffer conditions. (D) Samples representing the 32-min time point from panel B were detected with anti-SAK polyclonal antibodies (α-SAK), anti-hirudin polyclonal antibodies (α-Hir), and anti-hirudin C-terminal specific monoclonal antibody MAT 108 (α-C-Hir).