FIG. 4.
Expression and secretion assays of hEPO and tGH hybrids in E. coli C600. The hybrids were visualized by SDS–12% PAGE followed by a Western blot assay revealed with anti-protease B polyclonal antibodies. Cultures were induced for 4 h at 37°C with 1 mM IPTG after the bacterial cultures reached an OD600 of 1.0. Ten-microliter samples of lysates (L) and supernatants (S) that were concentrated 10 times with respect to the lysates by 10% TCA precipitation were loaded in the gel. Std, Benchmark prestained protein ladder. (A) Expression of hybrid hEPO-93CTPB. Lanes: 1 and 2, clone harboring pSE420 plus pRUW4; 3 and 4, clone with phEPO-93CTPB; 5 and 6, clone with phEPO-93CTPB plus pRUW4. (B) Expression of hybrid tGH2-93CTPB. Lanes: 1 and 2, clone with pSE420 plus pRUW4; 3 and 4, clone with ptGH2-93CTPB; 5 and 6, clone with ptGH2-93CTPB plus pRUW4. The arrows indicate the electrophoretic migration of the protein bands expected in each case. (C) Expression of hybrid hEPO-181CTPB. Lanes: 1 and 2, clone 3-1 (phEPO-181CTPB plus pRUW4 in E. coli DH5α); 3 and 4, clone 12-1 (phEPO-181CTPB plus pRUW4 in E. coli DH5α); 5 and 6, clone 4-1 (phEPO-181CTPB plus pRUW4 in E. coli DH5α). (D) Lanes: 1 and 2, clone 6-1 (ptGH-181CTPB plus pRUW4 in E. coli DH5α); 3 and 4, clone 5-1 (ptGH-181CTPB plus pRUW4 in E. coli DH5α); 5 and 6, clone 2-1 (ptGH-181CTPB plus pRUW4 in E. coli DH5α).