A versatile phage lambda expression vector system for cloning in Escherichia coli.
By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.
Sieg
K
K
Institut für Genetik der Universität zu Köln, F.R.G.
Kun
J
J
Pohl
I
I
Scherf
A
A
Müller-Hill
B
B
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015951:Factor Xa; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D004247:DNA; D001616:beta-Galactosidase; D012697:Serine Endopeptidases; D015951:Factor Xa