Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system.
A new bacterial vector was constructed that combines the attractive features of gene fusion vectors and phagemids. A gene of interest cloned into this new vector can either be expressed as fusion protein or be prepared as single-stranded template DNA within the same system. Thus, time consuming subcloning procedures changing the bacterial vector according to the required method are avoided. As an example sequencing, expression and subsequent purification of site-directed mutants of herpes simplex virus type 1 thymidine kinase are discussed.
Michael
M
M
Department of Pharmacy, ETH Zurich, Switzerland.
Gerber
S
S
Fetzer
J
J
Folkers
G
G
D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology; D017912:Inovirus / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics
D004277:DNA, Single-Stranded; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D013937:Thymidine Kinase