The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over.

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.

Recombinant human microplasmin: production and potential therapeutic properties.

The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen ( micro Plg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin ( micro Pli) and equilibrated with 5 mmol L(-1) citrate, pH 3.

Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains.

Genetically altered mice may exhibit highly variable phenotypes due to the variation in genetic background, which can only be circumvented by generation of inbred, isogenic gene-targeted and control mice. Here we report that an embryonic stem (ES) cell culture medium conditioned by a rabbit fibroblast cell line transduced with genomic rabbit leukemia inhibitory factor allows efficient derivation and maintenance of ES cell lines from all of 10 inbred mouse strains tested, including some that were presumed to be nonpermissive for ES cell derivation (129/SvEv, 129/SvJ, C57BL/6N, C57BL/6JOla, CBA/CaOla, DBA/2N, DBA/1Ola, C3H/HeN, BALB/c, and FVB/N). Germline transmission was established by blastocyst injection of established ES cell lines after 10 or more passages from all of seven strains tested (129/SvJ, C57BL/6N, C57BL/6JOla, DBA/2N, DBA/1Ola, BALB/c, and FVB/N), by diploid aggregation of ES cell lines from all of four strains tested (129/SvEv, C57BL/6N, CBA/ CaOla, and FVB/N), or by tetraploid aggregation of ES cell lines from all of three strains tested (129/SvEv, C57BL/6N, and CBA/CaOla).

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

Background: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance.

Methods And Results: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20).

Recombinant staphylokinase variants with reduced antigenicity due to elimination of B-lymphocyte epitopes.

Site directed mutagenesis (350 variants) of recombinant staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activity or reduced binding of human staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, which absorbed down to less than a third of SakSTAR-specific antibodies.