Increased protein expression through improved ribosome-binding sites obtained by library mutagenesis.
This report describes a method whereby library mutagenesis combined with drug selection was used to generate unique and efficient ribosome-binding sites (RBS) for expressing recombinant proteins in Escherichia coli. The RBS was deleted from a vector expressing beta-lactamase and replaced with a 16-base sequence containing a library of mutations. Selection of the library with ampicillin yielded several unique RBS sequences that were more efficient than ompA RBS for expressing a bacterial (beta-lactamase) and a mammalian protein (single-chain Fv antibody). The described approach provides a practical means to improve recombinant protein expression and, also, provides new sequences to further evaluate the complex regulatory mechanism underlying translation initiation.
Wilson
B S
BS
Hybritech Incorporated, San Diego, CA.
Kautzer
C R
CR
Antelman
D E
DE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D018014:Gene Transfer Techniques; D007128:Immunoglobulin Fragments / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012270:Ribosomes / Q000378:metabolism*; D001618:beta-Lactamases / Q000235:genetics
D007128:Immunoglobulin Fragments; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D001618:beta-Lactamases