Recombinant strains and medium formulation for enhancing secretion titer using a type III secretion system
The present disclosure provides a recombinant Salmonella strain having a Type III secretion system (T3SS) comprising mutation which enhance protein expression and production. Additionally, methods and kits for using the recombinant Salmonella strain for producing a protein of interest are provided. Additionally, an optimized medium that increases protein expression in a Salmonella strain having a Type III secretion system (T3SS) is provided.
1 . A recombinant Salmonella strain having a Type III secretion system (T3SS) comprising
(a) an insertion of a protein coding sequence between the hill coding sequence and the hilD 3′UTR suitable to increase hilD expression in the strain; or
(b) an insertion of a protein coding sequence between the hill coding sequence and the hilD 3′UTR suitable to increase hilD expression in the strain; and a deletion of hilE;
wherein the protein coding sequence encodes a B-barrel protein.
2 . The recombinant Salmonella strain of claim 1 , wherein the strain is enterica serovar Typhimurium LT2.
3 . The recombinant Salmonella strain of claim 1 , wherein the strain comprises (b).
4 . The recombinant Salmonella strain of claim 1 , wherein the recombinant Salmonella strain has at least 4 fold increased secretion titer of a protein of interest by the Type III secretion system as compared to a non-recombinant Salmonella strain having a Type III secretion system.
5 . The recombinant Salmonella strain of claim 1 , wherein the strain further comprises at least one modification to reduce the pathogenicity or virulence of the Salmonella strain, wherein the at least one modification comprises a deletion of at least one of sipB, sipC, sipD, flhDC, pathogenicity island 4 (SPI-4), and pathogenicity island 5 (SPI-5).
6 . The recombinant Salmonella strain of claim 1 , wherein the protein coding sequence comprises at least 270 base pairs.
7 . The recombinant Salmonella strain of claim 6 , wherein the wherein the protein coding sequence comprises at least 700 base pairs.
8 . The recombinant Salmonella strain of claim 5 , wherein the at least one gene is sipD.
9 . The recombinant Salmonella strain of claim 3 , wherein the strain is a hilE gene knockout.
10 . The recombinant Salmonella strain of claim 1 , wherein the β-barrel protein is a fluorescent protein.
11 . An in vitro method of making a heterologous protein of interest, the method comprising
(a) expressing a heterologous protein of interest in the recombinant Salmonella strain of claim 1 , wherein the protein of interest is fused to a targeting polypeptide that directs the protein to the Type Ill secretion system (T3SS) of the strain, and wherein the protein of interest is secreted into the medium in which the recombinant Salmonella strain is suspended.
12 . The in vitro method of claim 11 , wherein the method comprises introducing a vector encoding the heterologous protein of interest into the recombinant Salmonella strain before (a).
13 . The method of claim 11 , wherein the targeting polypeptide is an N-terminal secretion tag under control of a promoter that participates in the activation signal cascade for T3SS.
14 . The method of claim 11 , wherein the heterologous protein of interest comprises a cleavable linker between the targeting polypeptide and the protein of interest.
15 . The method of claim 11 , wherein the method further comprises
(b) isolating the heterologous protein of interest from the medium.
16 . The method of claim 15 , wherein the method further comprises
(c) contacting the heterologous protein of interest with an enzyme capable of cleaving the protein of interest from the targeting tag.
17 . The method of claim 12 , wherein only one vector is introduced into the recombinant Salmonella strain.
18 . The method of claim 11 , wherein the recombinant Salmonella strain is cultured in a defined medium able to increase protein secretion comprising
about 10 g/L peptone,
about 5 g/L yeast extract,
glycerol or glucose in an amount effective to increase the osmolality of the medium,
about 80 mM to about 120 mM potassium phosphate pH 7.4; and
optionally about 150 mM to about 210 mM NaCl.
19 . The method of claim 18 , wherein the defined medium comprises about 0.4% w/v glycerol.
20 . An in vitro culture comprising
(a) the recombinant Salmonella strain of claim 1 ; and
(b) defined culture medium for increasing protein secretion comprising:
about 10 g/L peptone,
about 5 g/L yeast extract,
glycerol or glucose in an amount effective to increase the osmolarity of the medium,
about 80 mM to about 120 mM potassium phosphate pH 7.4, and optionally about 150 mM to about 210 mM NaCl.
21 . The in vitro culture of claim 20 , wherein the defined medium comprises about 0.2 to about 0.6% w/v glycerol or glucose in an amount effective to increase the osmolarity of the medium.
22 . A kit for making a protein of interest comprising
(a) the recombinant Salmonella strain of claim 1 ; or
(b) an in vitro culture comprising
(i) the recombinant Salmonella strain of claim 1 ; and
(ii) defined culture medium for increasing protein secretion comprising:
about 10 g/L peptone,
about 5 g/L yeast extract,
glycerol or glucose in an amount effective to increase the osmolality of the medium,
about 80 mM to about 120 mM potassium phosphate pH 7.4; and
optionally about 150 mM to about 210 mM NaCl.