Process for preparing variant of
View Patent ↗A variant of Erysipelothrix rhusiopathiae surface protective antigen SpaA protein or of a shortened form of SpaA (ΔSpaA) in which a portion of SpaA protein is deleted for protection from Erysipelothrix rhusiopathiae infection and a process for preparing the same are provided. Introduction of amino acid substitution at a specific site in the amino acid sequence of SpaA or ΔSpaA protein provides a variant of SpaA or ΔSpaA protein which is immunogenic and is expressed in E. coli as inclusion bodies. The variant of SpaA or ΔSpaA protein of the present invention may easily be recovered and purified since it is expressed in E. coli as inclusion bodies.
1. A process for screening mutant proteins of Erysipelothrix rhusiopathiae surface protective antigen SpaA protein, or of a shortened ΔSpaA form thereof, for a variant protein having immunogenicity and being expressed in Escherichia coli as inclusion bodies, comprising:
mutating a gene coding for said SpaA or ΔSpaA protein so that amino acid substitution may be introduced in the amino acid sequence of said SpaA or ΔSpaA protein;
allowing the resulting mutated gene to be expressed in E. coli ; and
screening the mutant proteins for a variant protein that formed inclusion bodies.
2. The process of claim 1 , which comprises the following steps (A) to (D):
(A) introducing mutation in a gene coding for soluble Erysipelothrix rhusiopathiae surface protective antigen SpaA or ΔSpaA protein so that amino acid substitution may be introduced;
(B) transforming E. coli cells with an expression vector containing the resulting mutated gene;
(C) screening transformed E. coli cells expressing mutant proteins of SpaA or ΔSpaA protein for a variant protein forming insoluble inclusion bodies in the transformed E. coli cells; and
(D) culturing E. coli cells expressing the variant protein that forms insoluble inclusion bodies for recovery of the inclusion bodies within the cells.
3. The process of claim 2 , further comprising, after step (D), the following steps (E) to (F):
(E) administering the inclusion bodies or the inclusion bodies treated with a solubilizing agent to an animal sensitive to Erysipelothrix rhusiopathiae infection and then challenging said animal with a virulent strain of Erysipelothrix rhusiopathiae ; and
(F) observing survival or death of the animal sensitive to Erysipelothrix rhusiopathiae to thereby assess the presence of a protective activity against Erysipelothrix rhusiopathiae infection.
4. The process of any one of claims 1 to 3 , wherein said amino acid substitution is in the SpaA protein comprising the amino acid sequence encoded by SEQ ID NO:7 and is one or a combination of more than one selected from the group consisting of (1) to (7) as described below:
(1) amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 69 of SEQ ID NO:2;
(2) amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 154 of SEQ ID NO:2;
(3) amino acid substitution of isoleucine to threonine at the residue corresponding to residue position 203 of SEQ ID NO:2;
(4) amino acid substitution of histidine to glutamine at the residue corresponding to residue position 214 of SEQ ID NO:2;
(5) amino acid substitution of methionine to threonine at the residue corresponding to residue position 253 of SEQ ID NO:2;
(6) amino acid substitution of aspartic acid to glycine at the residue corresponding to residue position 278 of SEQ ID NO:2; and
(7) amino acid substitution of arginine to glycine at the residue corresponding to residue position 531 of SEQ ID NO:2.
5. The process of any one of claims 1 to 3 , wherein said amino acid substitution is in the SpaA protein comprising the amino acid sequence encoded by SEQ ID NO:7 and is one selected from the group consisting of (a) to (h) as described below:
(a) amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 69 of SEQ ID NO:2;
(b) amino acid substitution of isoleucine to threonine at the residue corresponding to residue position 203 of SEQ ID NO:2;
(c) amino acid substitution of histidine to glutamine at the residue corresponding to residue position 214 of SEQ ID NO:2;
(d) amino acid substitution of aspartic acid to glycine at the residue corresponding to residue position 278 of SEQ ID NO:2;
(e) amino acid substitution of arginine to glycine at the residue corresponding to residue position 531 of SEQ ID NO:2;
(f) amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 154 of SEQ ID NO:2 and amino acid substitution of isoleucine to threonine at the residue corresponding to residue position 203 of SEQ ID NO:2;
(g) amino acid substitution of histidine to glutamine at the residue corresponding to residue position 214 of SEQ ID NO:2 and amino acid substitution of methionine to threonine at the residue corresponding to residue position 253 of SEQ ID NO:2; and
(h) amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 69 of SEQ ID NO:2, amino acid substitution of glutamic acid to glycine at the residue corresponding to residue position 154 of SEQ ID NO:2, and amino acid substitution of isoleucine to threonine at the residue corresponding to residue position 203 of SEQ ID NO:2.
6. The process of any one of claims 1 to 3 , wherein said Erysipelothrix rhusiopathiae is selected from the group consisting of Fujisawa strain, Koganai strain, Tama 96 strain, SE-9 strain and Shizuoka 63 strain.
7. The process of claim 1 , wherein the SpaA or ΔSpaA protein before introduction of said amino acid substitution has, respectively, the amino acid sequence of SEQ ID NO:2 or the sequence of SEQ ID NO:2 in which the C-terminal 206 amino acid residues are deleted.