Purification, characterization and biological evaluation of recombinant leech-derived tryptase inhibitor (rLDTI) expressed at high level in the yeast Saccharomyces cerevisiae.

Abstract

An efficient expression/purification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUC2). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI. Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular-mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of approximately 0.1 nM and approximately 1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.