Co-expression of recombinant proteins
a recombinant protein and co-expression technology, applied in the field of molecular biology, biochemistry and vaccinology, can solve the problems of increased production time and cost, and increased production cost, and achieves stable proteolytic activity
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example 1
[0063] This Example describes the construction of plasmid JB-3120-2, which contains the T7 H91A hin47 gene encoding the endogenous leader sequence. The procedure employed is shown schematically in FIG. 1A.
[0064] The production of the mature recombinant H91A Hin47 protein from E. coli has been described in the aforementioned U.S. Pat. No. 5,506,139. This protein was produced at 40 to 50% of total protein in a soluble form. The bacterial HtrA proteins are located in the periplasmic membrane and may function as chaperones if located there. In order to direct the mutant, non-proteolytic H91A Hin47 protein to the periplasmic membrane, the endogenous leader was added. Plasmid DS-2140-3 is a pBR328-based plasmid that contains the T7 H91A hin47 gene cassette between EcoR I and Cla I sites (FIG. 1A). Plasmid JB-1 172-2-5 is a pUC-based plasmid that contains the wild-type htrA gene with 5′- and 3′-flanking sequences. Plasmid JB-1172-2-5 was digested with Cla I and Sal I and the 0.6 kb 3′-fla...
example 2
[0065] This Example describes the production and purification of recombinant H91A Hin47 protein with its endogenous leader sequence.
[0066] Cells were grown at 37° C. in NZCYM medium using the appropriate antibiotic selection to A578 of 0.3 before addition of lactose to 1.0% for 4 hours. Samples were adjusted to 0.2 OD / μl with SDS-PAGE lysis+loading buffer and the same amount of each protein sample was loaded onto SDS-PAGE gels (ref 18). The mature H91A Hin47 protein was produced at ˜50% of total protein, while the H91A Hin47+leader protein was produced at 20 to 25% of total protein (FIG. 2).
[0067] The purification of the mature soluble H91A Hin47 protein has been described in U.S. Pat. No. 5,506,139. The H91A Hin47 +leader protein was found to be associated with the pellet after two extractions of E. coli cells with 50 mM Tris-HCl, pH 8.0 and 50 mM Tris-HCl, pH 8.0 containing 0.5% Triton X-100 and 10 mM EDTA. The pellet containing H91A Hin47 was solubilized in 50 mM Tris-HCl, pH 8...
example 3
[0069] This Example illustrates the construction of plasmid DS-2342-2-2, which contains the T7 H91A hin47, T7 V38 hia, and E. coli cer genes. The procedure employed is shown schematically in FIG. 5.
[0070] Plasmid DS-1872-2-2 is a pBR328-based vector containing a 2.2 kb EcoR I T7 H91A hin47 gene cassette (FIG. 5). Plasmid BK-96-2-11 is a pBR328-based vector that contains a T7 V38 hia gene cassette, the E. coli cer gene, and a kanamycin resistance gene; and this plasmid has been described in the aforementioned U.S. patent application Ser. No. 09 / 268,347. BK-96-2-11 was linearized by digestion with EcoR I, dephosphorylated, and the EcoR I T7 H91A hin47 gene fragment inserted, to generate plasmid DS-2342-2-2, that co-expresses the H91A hin47 and V38 hia genes. This plasmid thus contains tandem T7 H91A hin47 and T7 V38 hia genes in the same orientation. Plasmid DNA was introduced into electrocompetent E. coli BL21(DE3) cells using a BioRad electroporator, and recombinant E. coli strain ...
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