CONTROL SYSTEMS FOR RECOMBINANT MANIPULATIONS.

摘要

Two types of convenient portable control cassettes for the expression of protein encoding sequences in procaryotes. Both cassettes comprise the PL promoter from lambda phage, which is controllable by means of a temperature sensitive repressor, operably linked to the ribosome binding site for N-gene (NRBS). The cassette is either bordered by restriction sites upstream of the PL promoter and immediately downstream from the NRBS permetting the insertion of a desired sequence containing its own start codon or contains an ATG start codon within the cassette and has a restriction site immediately 3' of the start codon. A novel universal dominant selectable marker cassette is also disclosed. The marker comprises the coding sequences for aminoglycoside phosphotransferase I(APH-I) which has been modified and truncated so as to render its use in recombinant vectors more convenient. The modified, truncated sequence (mtAPH-I) gene is capable, upon expression, of conferring resistance to a number of antibiotics on the host. One of these antibiotics, G418, is toxic to eucaryotic as well as procaryotic hosts. Also disclosed are fusion proteins having N-terminal sequences corresponding to a desired peptide sequence, and C-terminal sequences comprising the amino acids encoded by mtTAPH-I. These proteins are dominant selectable fusion flags which are useable in a wide variety of lacY<-> hosts. Such fusion flags permit direct selection of transformants, diagnosis of expression levels for desired seqquences, and may afford a method to purify the attached desired sequences.