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The bacterial production of recombinant rat calpain II has been improved greatly by the use of two compatible plasmids for the two subunits. The calpain small subunit C-terminal fragment (21 kDa) was expressed from a new A15-based vector created by cloning T7 control elements into pACYC177. This vector is compatible with the ColE1-based pET-24d(+) vector containing the calpain large subunit, and the yield of calpain activity was increased at least 16-fold by co-expression from these two vectors. A high level of activity was also obtained from a bicistronic construct containing both subunit cDNAs under the control of one T7 promoter. The addition of a C-terminal His-tag to the large subunit simplified purification without affecting subunit association or enzyme activity. The active-site cysteine 105 was mutated to alanine, causing complete loss of activity. The yield of purified C105A-calpain II (80 + 21 kDa) dimer following three column chromatography steps was 10 mg/l of cell culture. This provides a purified calpain, stable to autolysis and oxidation, which is likely to facilitate crystallization in both the presence and absence of calcium.

A 4.3-kDa variant of Type I antifreeze protein (AFP9) was purified from winter flounder serum by size exclusion chromatography and reversed-phase HPLC. By the criteria of mass, amino acid composition, and N-terminal sequences of tryptic peptides, this variant is the posttranslationally modified product of the previously characterized AFP gene 21a. It has 52 amino acids and contains four 11-amino acid repeats, one more than the major serum AFP components. The larger protein is completely a-helical at 0 "C, with a melting temperature of 18 "C.It is considerably more active as an antifreeze than the three-repeat winter flounder AFP and the four-repeat yellowtail flounder AFP, both on a molar and a mg/mL basis. Several structural features of the four-repeat winter flounder AFP, including its larger size, additional ice-binding residues, and differences in ice-binding motifs might contribute to its greater activity. Its abundance in flounder serum, together with its potency as an antifreeze, suggest that AFP9 makes a significant contribution to the overall freezing point depression of the host.

No abstract

The 9-kDa, Thr-, Ser-, and Cys-rich thermal hysteresis protein from spruce budworm (sbwTHP) is 10Ϫ30 times more effective than fish antifreeze proteins (AFPs) at depressing solution freezing points via ice-crystal growth inhibition. Since this insect protein is only available in microgram quantities from its natural source, recombinant sbwTHP was produced from inclusion bodies in Escherichia coli by a refolding protocol. Incompletely folded forms were removed during ion-exchange and reverse-phase chromatography, resulting in fully active sbwTHP that was indistinguishable in its properties from native sbwTHP. The antifreeze was completely inactivated by reduction, showed no reaction with sulfhydryl reagents, and was not inhibited by EDTA. All eight cysteine residues appear to be involved in disulfide bond formation. Tryptic cleavage and peptide analysis is consistent with linkages between the first and second cysteine residues, the third and fourth, fifth and eighth, and the sixth and seventh. NMR analysis confirmed that the fully folded form of sbwTHP was well structured and had a single conformation. Both NMR and CD spectra indicate the presence of extensive β structure (70Ϫ80 %) with little or no A helix. The protein maintains antifreeze activity over a broad range of pH values, and its conformation is independent of both temperature (over the range 0°C to 20°C), and the presence of 50% trifluoroethanol.Keywords : CD; disulfide linkages; NMR; protein folding; thermal hysteresis.Antifreeze proteins (AFPs), traditionally called thermal hysteresis proteins (THPs) in the insect literature, have recently been purified and characterized from two unrelated insect species, spruce budworm (Choristoneura fumiferana), and the common yellow mealworm (Tenebrio molitor) [1,2]. Their ability to depress the non-equilibrium freezing point of a solution below its melting point (thermal hysteresis activity) is exceptional in two respects. One is that the freezing point depression (4Ϫ5°C) measured in these insects is several degrees greater than that seen with AFPs from other organisms such as fish and plants [3,4]. The other is that their specific thermal hysteresis activity (freezing point depression as a function of concentration) is at least an order of magnitude greater than that obtained with fish AFPs [5,6]. Despite their superior activity, insect THPs operate at the ice/water interface to control ice crystal growth and shape and, therefore, appear to function by the adsorption-inhibition mechanism originally described for fish AFPs [7].The recently described spruce budworm (moth) and mealworm (beetle) THPs are present in larval hemolymph. Both are small, Cys-, Thr-, and Ser-rich proteins of 8Ϫ9 kDa that are extensively disulfide bonded. Despite these physical resemblances and their similar hyperactivity, they show no obvious homology at the DNA or amino acid sequence level, and the spacing of their numerous cysteine residues is quite different. Moreover, the mealworm THP is clearly built up of 12-aminoacid repeats, wherea...