Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.
A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.
Auerswald
E A
EA
Abteilung für Klinische Chemie und Klinische Biochemie, Klinikum Innenstadt, Ludwig-Maximilians-Universität München, Germany.
Morenweiser
R
R
Sommerhoff
C P
CP
Piechottka
G P
GP
Eckerskorn
C
C
Gürtler
L G
LG
Fritz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D007525:Isoelectric Focusing; D007865:Leeches / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D014361:Trypsin Inhibitors / Q000096:biosynthesis* / Q000737:chemistry; D014779:Virus Replication / Q000187:drug effects*
D011506:Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C090901:leech-derived tryptase inhibitor C