1 / 10

pHCE vector

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.

milly
Download Presentation

pHCE vector

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. pHCE vector Insun Song

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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).

  7. 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.

  8. 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.

  9. 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.

  10. Comparison of production

More Related