Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.
A high-copy-number plasmid expression vector (pIJ6021) was constructed that contains a thiostrepton-inducible promoter, PtipA, from Streptomyces lividans 66. The promoter and ribosome-binding site of tipA lie immediately upstream from a multiple cloning site (MCS) which begins with a NdeI site (5'-CATATG) that includes the tipA translational start codon (ATG), allowing the synthesis of native proteins. Transcriptional terminators occur just upstream from PtipA and immediately downstream from the MCS. To demonstrate the utility of pIJ6021, two streptomycete genes and a growth hormone-encoding gene from flounder (Paralichthys olivaceus) were cloned in the vector and expressed in S. lividans or S. coelicolor A3(2). A derivative of pIJ6021, pIJ4123, has a unique NdeI site positioned downstream from a nucleotide sequence that encodes a His6 sequence and thrombin cleavage site. pIJ4123 can be used to produce His-tagged fusion proteins that can be readily purified by Ni(2+)-affinity chromatography; if necessary, the His6 tag can be removed by digestion with thrombin. The vectors contain a kanamycin-resistance-encoding gene for the selection of transformants.
Takano
E
E
John Innes Centre, Colney, Norwich, UK.
White
J
J
Thompson
C J
CJ
Bibb
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*; D013883:Thiostrepton / Q000494:pharmacology; D014170:Transformation, Genetic
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006639:Histidine; D013883:Thiostrepton