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RpoDhs-lacZ)) (27), which is a MC4100 derivative that lacks the rpoH gene but is able to grow at 37 due to the overproduction of GroEL and GroES, was used for the purification of 32. AD202 (MC4100 ompT::kan) (28) was used for the purification of FtsH. HB101 (supE (mcrC-mrr) recA ara-14 proA lacY galK rpsL xyl-5 mtl-1 leuB thi-1) and MC4100 were used for the purification of DnaK and DnaJ, respect
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[21] has not been so encouraging . the antigenic/therapeutic protein in vivo[21] has not been so encouraging . the antigenic/therapeutic protein in vivo and to stimulate [25] potent specific humoral and cellular immune responses . RNA viral vectors, such as retrovirus and lentivirus, allow long-term expression of the transgene, while DNA viral vectors allow expression in episomal form. Viral
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Derivatives (26). The nucleotide sequences of all of the PCRamplified and cloned fragments were confirmed by DNA sequence analysis. Media and Chemicals--L broth (31) was used for growing cells; ampicillin (50 g/ml) was added when necessary. Chemicals were purchased from Nacalai Tesque (Kyoto, Japan), Wako Pure Chemicals (Osaka, Japan), or Sigma-Aldrich Japan K.K. (Tokyo, Japan). Protein Purificati
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Derivatives (26). The nucleotide sequences of all of the PCRamplified and cloned fragments were confirmed by DNA sequence analysis. Media and Chemicals--L broth (31) was used for growing cells; ampicillin (50 g/ml) was added when necessary. Chemicals were purchased from Nacalai Tesque (Kyoto, Japan), Wako Pure Chemicals (Osaka, Japan), or Sigma-Aldrich Japan K.K. (Tokyo, Japan). Protein Purificati
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Ases its substrates. After dissociation from Hsp70, the polypeptide chains fold into a functional three-dimensional architecture. A model has been proposed in which Hsp40 first recognizes and binds substrate polypeptides and then transfers them to Hsp70. However, in some cases, DnaK itself recognizes and binds to substrate polypeptides independent of DnaJ, as in the case of 32 (24). Because most o
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K authentic promoters were amplified byPCR and cloned under the trc promoter. In the case of DnaKHis and DnaJ-His, six histidine codons were added to the 3 -end of the corresponding genes on the pKV1142 derivatives. Both DnaK-His and DnaJ-His have two extra amino acids, arginine and serine, between the last amino acid residue of the intact protein and the His tag, which is derived from the BglII s