pHCE vector Insun Song
Conventional vector • E. coli expression vector (including a trp-lac fusion promoter) can be effectively induced by IPTG, is the most frequently used system. • Chemical, thermal inducer have problem.
pHCE vector • Constitutive promoterfacilitates the high-level expression of foreign proteins without induction by chemical inducers or heat • D-AAT(from Geobacillus toebii) could be efficiently expressed in recombinant E. coli in the absence of the expression vectors pUC18 and pTrc99A. • promoter of D-AAT, named pHCE(high constitutive expression), this studyhave developed a constitutive expression vector using pHCE and attempted the high-level production of the TNF-α in recombinant E. coli by cloning the TNFα gene into the pHCE vector. • Tumor necrosis factor α (TNF α) -multipotent cytokine -induction of cellular responses
Construction of pHCE-IIB Fig. 1. Construction of HCE expression vector, pHCE-IIB. HCE promoter, high-level constitutive expression promoter, Ampr, ampicillin resistance gene; ori, origin of replication ; MCS, multi-cloning site; rrnBT1T2, transcription terminator.
pET14b & pHCE-IIB • The expression efficiency of recombinant rhTNFα using pHCE-IIB was compared with that of the pET14B vector under the control of a T7 promoter induced by IPTG. • After digesting the amplified hTNF fragment with NcoI and BamHI, it was ligated to the NcoI/BamHI-cleaved pHCE- IIB and pET-14b vectors, named pHCE-IIB-hTNFα and PET14b-hTNFα
Expression of rhTNFα Fig. 2. SDS-PAGE of rhTNFα expressed by pHCE-IIB-hTNFα and pET14b-hTNFα. M, marker; lane 1, pHCE-IIB (negative control); lane 2, pHCE-IIB-TNFα (total); lane 3, pHCE-IIB-TNFα (insoluble); lane 4, pHCE-IIB-TNFα (soluble); lane 5, pET14b-TNFα(total); lane 6, pET14b-TNFα (insoluble); lane 7, pET14b-TNFα(soluble).
Fig. 5. SDS-PAGE of rhTNFα expressed by pHCE-IIB-hTNFα and pET14b-hTNFα relative to time. (A) rhTNFα expressed by host cells harboring pET14b-TNFα without (lanes 1–3) and with (lanes 4–6) IPTG induction after incubation for 19, 21, and 23 h. (B) rhTNFα expressed by host cells harboring pHCE-IIB-TNFα after incubation for 17, 19, 21, and 23 h (lanes 1–4). M, Marker.
Biological activity of rhTNFα Fig. 3. L929 cytotoxicity assay of rhTNFα. The cytotoxicity of the soluble rhTNFα produced from E. coli cells harboring pHCE-IIB-TNFα (●) or pET14b-TNFα with IPTG induction (▲) and standard rhTNFα (■) was determined by a colorimetric MTT assay.
Mass Production of rhTNFα DCW Fig. 4. Time course of recombinant E. coli growth when hosting pHCE-IIB-hTNFα (○) and pET14b-hTNFα with IPTG induction (△) and without IPTG induction (□). The recombinant E. coli was cultivated in 3 l of complex media (CPGY) in a 5 l fermenter.