Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli.

Abstract

The gene for the outer membrane lipoprotein (lpp), the most abundant protein of Escherichia coli, was used to construct multi-purpose expression cloning vehicles. These vehicles consist of two types in terms of gene expression, one for constitutive (pIN-I type; three vehicles) and the other for inducible (pIN-II type; three vehicles) gene expression, and have the following features: (a) The lpp gene was inserted into a multicopy plasmid, pBR322, and the tet gene was removed to keep the size of the vehicles minimal (approximately 5 kb). (b) A nucleotide sequence of 22 bp which contains EcoRI, HindIII, and BamHI sites was inserted at the position of the third amino acid of the prolipoprotein. (c) The same nucleotide sequence was also inserted in two other reading frames at the same position. There are no other EcoRI, HindIII, and BamHI sites in the vehicles. Therefore, six different types of restriction fragments (EcoRI-EcoRI, HindIII-HindIII, BamHI-BamHI, EcoRI-HindIII, EcoRI-BamHI, and HindIII-BamHI) can be cloned at this position in any of the three different reading frames. (d) The nucleotide sequence from position 46 to 168 of the lpp gene was deleted. However, the 3' end position of the lpp gene of 154 bp was retained, which contains not only translation termination codons in three different reading frames but also the transcription termination signal of the lpp gene. Thus, this sequence is assumed to prevent unnecessary translation as well as transcriptional read-through of a cloned gene.(ABSTRACT TRUNCATED AT 250 WORDS)

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Affiliated Institutions

• Mitsubishi Group (Japan) JP
• Institute of Life Sciences IN
• State University of New York US
• Stony Brook University US

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