Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo.
The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described.
Jespers
L
L
Laboratorie de Biochimie Physique et des Biopolymères, UCL, Louvain-la-Neuve, Belgium.
Sonveaux
E
E
Fastrez
J
J
Phanapoulos
A
A
Davison
J
J
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005814:Genes, Viral; D005822:Genetic Vectors; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics
D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D009113:Muramidase