Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.
We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein.
Department of Chemistry, Harvard University, Cambridge, MA 02138.
K D
KD
MacFerrin
M P
MP
Terranova
S L
SL
Schreiber
G L
GL
Verdine
D000595:Amino Acid Sequence; D001483:Base Sequence; D015704:CD4 Antigens / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D019914:Taq Polymerase
D015704:CD4 Antigens; D011506:Proteins; D011994:Recombinant Proteins; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase