The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.
Basic fibroblast growth factor (FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration, and survival of many cell types. The human FGF-2 gene (encoding residues 1-155) was synthesized by PCR from 20 oligonucleotides and cloned into plasmid pET-32a. A high expression level (1 g/liter) of a fused protein thioredoxin/FGF-2 was achieved in Escherichia coli strain BL21(DE3). The fusion protein was purified from the soluble fraction of cytoplasmic proteins on a Ni-NTA agarose column. After cleavage of the thioredoxin/FGF-2 fusion with recombinant human enteropeptidase light chain, the target protein FGF-2 was purified on a heparin-Sepharose column. The yield of FGF-2 without N- and C-terminal tags and with high activity was 100 mg per liter of cell culture. Mutations C78S and C96S in the amino acid sequence of the protein decreased FGF-2 dimer formation without affecting its solubility and biological activity.
Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer
stabilized in solution by the apolipoprotein or a special membrane scaffold
protein (MSP). In this work, the applicability of LPN-based membrane mimetics
in the investigation of water-soluble membrane-active peptides was studied. It
was shown that a pore-forming antimicrobial peptide arenicin-2 from marine
lugworm (charge of +6) disintegrates LPNs containing both zwitterionic
phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In
contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel
gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 :
1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs
containing zwitterionic lipids (POPC), and it weakly interacts with the protein
component of nanodiscs, MSP (charge of –6). The neurotoxin II (NTII,
charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine
receptor, shows a comparatively low affinity to LPNs containing anionic lipids
(POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The
obtained data show that NTII interacts with the LPN/POPC/DOPS surface in
several orientations, and that the exchange process among complexes with
different topologies proceeds fast on the NMR timescale. Only one of the
possible NTII orientations allows for the previously proposed specific
interaction between the toxin and the polar head group of phosphatidylserine
from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. №
7. P. 2089–2097). These results indicate that LPNs can be used in
structural and functional studies of water-soluble membrane-active peptides
(probably except pore-forming ones) and in studies of the molecular mechanisms
of peptide-membrane interaction.
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