Methods and materials relating to gene expression

Abstract

An expression cassette for expressing a nucleic acid of interest derived from the regulatory region of the methylenomycin gene cluster of the SCP1 plasmid of Streptomyces coelicolor A3(2), and related materials and methods.

1 .- 50 . (canceled) 
     
     
         51 . An expression product produced by a method comprising:
 (1) providing an expression system comprising an expression cassette for the expression of a nucleic acid of interest, said expression cassette including:
 a. at least one regulatory portion including:
 i. a first regulatory element which includes at least one of the nucleic acid sequences mmyR encoding an MmyR polypeptide, and mmfR encoding an MmfR polypeptide; 
 ii. a second regulatory element which includes the nucleic acid sequence mmfL encoding an MmfL polypeptide; and 
 iii. a promoter (the “repressible promoter”), the function of which is repressed by the expression product of the first regulatory element, that repression being alleviated or removed by a product, the production of which is conferred by the MmfL polypeptide, and 
 
 b. a heterologous nucleic acid of interest, in operative association with said promoter; and 
   (2) maintaining said expression system under conditions suitable for expression of the nucleic acid of interest.   
     
     
         52 . (canceled) 
     
     
         53 . A nucleic acid consisting essentially of at least one of the nucleic acid sequences mmyR, mmfP, mmfH, mmfL, mmfR, mmyT, mmyO and mmyG, optionally with at least one respective upstream region. 
     
     
         54 . A nucleic acid according to  claim 53 , wherein said at least one nucleic acid sequence is selected from the following:
 a. the mmyR nucleic acid sequence encoding an MmyR polypeptide having the amino sequence of SEQ ID NO: 12 (optionally excluding the first 6 listed amino acids), or a variant thereof;   b. the mmfR nucleic acid sequence encoding an MmfR polypeptide having the amino acid sequence of SEQ ID NO: 11, or a variant thereof;   c. the mmfL nucleic acid sequence encoding an MmfL polypeptide having the amino acid sequence of SEQ ID NO: 17, or a variant thereof;   d. the mmfP nucleic acid sequence encoding an MmfP polypeptide having the amino acid sequence of SEQ ID NO: 20, or a variant thereof;   e. the mmfH nucleic acid sequence encoding an MmfH polypeptide having the amino acid sequence of SEQ ID NO: 21, or a variant thereof;   f. the mmyT nucleic acid sequence encoding an MmyT polypeptide having the amino acid sequence of SEQ ID NO: 22 or a variant thereof;   g. the mmyO nucleic acid sequence encoding an MmyO polypeptide having the amino acid sequence of SEQ ID NO: 23, or a variant thereof;   h. the mmyG nucleic acid sequence encoding an MmyG polypeptide having the amino acid sequence of SEQ ID NO: 24, or a variant thereof; and   i. the mmyB nucleic acid sequence comprising an MmyB polypeptide having the amino acid sequence set out in EMBL AJ276673, or a variant thereof.   
     
     
         55 . A nucleic acid according to  claim 53 , wherein the upstream region comprises a promoter. 
     
     
         56 . A nucleic acid according to  claim 55 , wherein the promoter is selected from the group consisting of:
 a. the mmyTOG promoter comprising at least some of residues 5452 to 5675 of SEQ ID NO: 18, or a variant thereof;   b. the mmfLHP promoter comprising at least some of residues 4716 to 4909 of SEQ ID NO: 19, or a variant thereof;   c. the mmy . . . XCAPK promoter comprising at least some of the complement of residues 18892 to 19123 or 15404 to 15977 of EMBL AJ276673, or a variant thereof;   d. the mmyYF promoter comprising at least some of residues 18892 to 19123 of EMBL AJ276673;   e. the mmyBQE promoter comprising at least some of the complement of residues 18892 to 19123 of EMBL AJ276673;   f. the mmyR promoter comprising at least some of residues 7965 to 8132 of SEQ ID NO: 19 (optionally excluding residues 8113 to 8132), or a variant thereof;   g. the mmfR promoter comprises at least some of residues 4613 to 4806 of SEQ ID NO: 18, or a variant thereof; and   h. the mmfL promoter comprising at least some of residues 4716 to 4909 of SEQ ID NO: 19, or a variant thereof.   
     
     
         57 . (canceled) 
     
     
         58 . A polypeptide encodable by one of the nucleic acid sequences mmyR, mmfP, mmfH, mmfL, mmfR, mmyT, mmyO and mmyG. 
     
     
         59 . A polypeptide according to  claim 58  having an amino acid sequence as shown in one of  FIGS. 8   a  to  8   h , or a variant thereof. 
     
     
         60 . A vector including a nucleic acid according to  claim 53 . 
     
     
         61 . An expression system containing one or more nucleic acid according to  claim 53 . 
     
     
         62 . A method of producing a polypeptide encodable by one of the nucleic acid sequences mmyR, mmfP, mmfH, mmfL, mmfR, mmyT, mmyO and mmyG, the method comprising maintaining an expression system as defined in  claim 61  under conditions suitable for producing the polypeptide. 
     
     
         63 . A method comprising providing a polypeptide produced according to the method of  claim 62  and subjecting the polypeptide to one or more purification steps. 
     
     
         64 . A nucleic acid according to  claim 55 , wherein the promoter is at least one of an mmfL promoter and an mmfR promoter comprising a palindromic sequence having the half-site 5′-GG(T/C)CGGT(A/T) (T/C)G(T/G)-3′ (SEQ ID NO: 1), or a variant thereof having sequence identity at seven or more corresponding positions within the half-site. 
     
     
         65 . An expression cassette according to  claim 64 , wherein the palindromic sequence has the half-site 5′-GGAAGGTATTA-3′ (SEQ ID NO: 2), or a variant thereof.