TY - JOUR
T1 - Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.
AU - Takano, Eriko
AU - White, Janet
AU - Thompson, Charles J.
AU - Bibb, Mervyn J.
N1 - Relation: http://www.rug.nl/gbb/
date_submitted:2007
Rights: University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute
PY - 1995
Y1 - 1995
N2 - 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 Ni2+-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.
AB - 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 Ni2+-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.
KW - Streptomyces lividans
KW - His6 tag
KW - redD
KW - β-keto acyl synthase
KW - flounder growth hormone
KW - tipA
KW - Streptomyces coelicolor
U2 - 10.1016/0378-1119(95)00545-2
DO - 10.1016/0378-1119(95)00545-2
M3 - Article
VL - 166
JO - Gene
JF - Gene
IS - 1
ER -