Inhibitorily active recombinant human stefin B. Gene synthesis, expression and isolation of an inhibitory active MS-2 pol-stefin B fusion protein and preparation of Des[Met1,2(2)]stefin B.
A synthetic gene coding for the human intracellular cysteine proteinase inhibitor, stefin B, was constructed from 13 chemically synthesized oligonucleotides according to the method of Khorana. The gene was inserted into the plasmid vector pTZ, amplified and sequenced. For expression, a temperature-inducible system producing fusion proteins was used. With the vector pEx31A containing the synthetic cystatin B gene, E. coli strain 537 produced a fusion protein of the N-terminal part of bacteriophage MS-2 polymerase and [Met-2Gly-1]stefin B. Lysates of the induced bacteria were inhibitorily active against papain. The fusion protein was expressed in high yield (about 20% of total E. coli proteins) and mostly deposited as inclusion bodies. The unfolded fusion protein was partially purified in the presence of urea. After refolding, approx. 6% of the protein was inhibitorily active against papain, human cathepsin H and B. Des[Met1,2(2)]stefin B was released by cyanogen bromide cleavage of the fusion protein and identified by N-terminal amino-acid sequence analysis. The non-separated cleavage products were also inhibitorily active after refolding. The estimated inhibition constants for the fusion protein and its cleavage products were similar to those reported for natural stefin B.
Thiele
U
U
Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München.
Auerswald
E A
EA
Gebhard
W
W
Assfalg-Machleidt
I
I
Popović
T
T
Machleidt
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D055313:Cystatin B; D015891:Cystatins; D004262:DNA Restriction Enzymes; D005796:Genes; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011480:Protease Inhibitors / Q000235:genetics*; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology
C524994:CSTB protein, human; D015891:Cystatins; D011480:Protease Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; C059239:cystatin B, des(Met(2)(1,2))-; D055313:Cystatin B; D004262:DNA Restriction Enzymes