Mapping domains in proteins: dissection and expression of Escherichia coli adenylyl cyclase.
We have used the pRE expression vector containing the Escherichia coli adenylyl cyclase gene (cya) with the unique NdeI restriction site CATATG at the initiation codon in conjunction with a family of self-complimentary oligonucleotides to create amino- and carboxyl-terminal domains in adenylyl cyclase. The three sets of oligonucleotides contain a TAA translation stop codon in all reading frames flanked by the NdeI restriction endonuclease sequence and one or two nucleotides (5' NNCATATGTTAATTAATTAACATATGNN 3'). Ligation of one of these annealed oligonucleotides into a restriction site or creation of 5' TAA/CATATG 3' translation stop/NdeI restriction site along a gene in the pRE expression vector facilitates the premature termination of protein synthesis thus yielding amino-terminal domains. Removal of a fragment of the gene corresponding to the amino-terminal portion by NdeI restriction and ligation brings the 3' end of the gene in frame with the initiator ATG. With this strategy, expression of the carboxyl-terminal domain of a protein is possible which is otherwise not as simple as the expression of the amino-terminal domain. The feasibility of expression of any domain of a protein is demonstrated using the cya gene to create several amino- and carboxyl-terminal domains of adenylyl cyclase.
Reddy
P
P
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA.
Hoskins
J
J
McKenney
K
K
D000262:Adenylyl Cyclases / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018387:Codon, Initiator; D004926:Escherichia coli / Q000201:enzymology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D015183:Restriction Mapping
D018387:Codon, Initiator; D009841:Oligonucleotides; D000262:Adenylyl Cyclases