Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A.
A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.
Kaji
H
H
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Minami-osawa, Hachioji-shi, Tokyo, 192-0397, Japan.
Yada-Wakatabe
R
R
Uehira
T
T
Terai
M
M
Takeda
A
A
Satoh
T
T
Samejima
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics; D012867:Skin / Q000378:metabolism*
D015891:Cystatins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins