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, p(tipa), 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 P-tipA 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 His(6) 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 His(6) tag can be removed by digestion with thrombin. The vectors contain a kanamycin-resistance-encoding gene for the selection of transformants.