TY - JOUR
T1 - Metallothioniens with interdomain hinges expanded by insertion mutagenesis
AU - Rhee, In Koo
AU - Lee, K. S.
AU - Huang, P. C.
N1 - Funding Information:
We thank Mrs Betty Chinn Smith, Mary Cismowslri and Mark Chemaik for their technical assistance and valuable comments in various phases of this work. A preliminary account of this study was presented earlier (Cismowslri et al., 1988). This study was supported in part by US NIH grant GM32606 to P.C.H. I.-K.R. is on sabbatical leave from the Department of Agricultural Chemistry, Kyungpook National University, Taegu, Korea and K.S.L. is the recipient of a Predoctoral Fellowship at Johns Hopkins University from Korean Ministry of Education.
PY - 1990/1
Y1 - 1990/1
N2 - Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.
AB - Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser. These interdomain expansions were made to test if such structural alterations would affect MT function. These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2. The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted. Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium. As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently. The level of activity, however, diminished with the length of the insert. Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent.
KW - Domain spacing
KW - Insertion mutagenesis
KW - Interdomain hinges
KW - Metallothionein
KW - Thiolate-metal cluster
UR - http://www.scopus.com/inward/record.url?scp=0025213047&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025213047&partnerID=8YFLogxK
U2 - 10.1093/protein/3.3.205
DO - 10.1093/protein/3.3.205
M3 - Article
C2 - 2184435
AN - SCOPUS:0025213047
SN - 1741-0126
VL - 3
SP - 205
EP - 213
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 3
ER -