PMID- 24275638
OWN - NLM
STAT- MEDLINE
DCOM- 20141008
LR  - 20180823
IS  - 1096-0279 (Electronic)
IS  - 1046-5928 (Linking)
VI  - 95
DP  - 2014 Mar
TI  - Development of lentiviral vectors for transient and stable protein overexpression 
      in mammalian cells. A new strategy for recombinant human FVIII (rhFVIII) 
      production.
PG  - 50-6
LID - S1046-5928(13)00242-8 [pii]
LID - 10.1016/j.pep.2013.11.005 [doi]
AB  - BACKGROUND: Recombinant protein overexpression in mammalian cells constitutes a 
      real challenge in therapeutic protein production. Following the discovery of 
      intron functionality in gene expression, various expression vectors that include 
      them in their sequences have been developed. In this study, the main goal was to 
      develop new lentiviral vectors (LVs) carrying different promoter and 
      intron-containing 5'UTR (5' untranslated region) combinations and the design of 
      LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. RESULTS: By 
      combining the human cytomegalovirus (CMV) or the elongation factor 1alpha (EF-1alpha) 
      promoters along with different 5'UTRs that included leader introns, between 2 and 
      12-fold increases were reached, when transient and stable expression of the 
      enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new 
      LVs provided with promoters and 5'UTRs from high expression genes, according to a 
      gene database, were designed. Three of them were shown to be superior to the 
      EF-1alpha promoter in three widely used cell lines. CONCLUSION: In the present work, 
      LVs containing different promoters and 5'UTRs were designed. In transient and 
      stable assays some of these constructs have shown higher activity compared with 
      commercial promoters and, therefore, constitute promising candidates for 
      therapeutic protein production.
CI  - Copyright (c) 2013 Elsevier Inc. All rights reserved.
FAU - Mufarrege, Eduardo Federico
AU  - Mufarrege EF
AD  - Laboratorio de Cultivos Celulares, Facultad de Bioquimica y Ciencias Biologicas, 
      Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 
      242, S3000ZAA Santa Fe, Argentina. Electronic address: mufarrege@fbcb.unl.edu.ar.
FAU - Antuna, Sebastian
AU  - Antuna S
AD  - Laboratorio de Cultivos Celulares, Facultad de Bioquimica y Ciencias Biologicas, 
      Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 
      242, S3000ZAA Santa Fe, Argentina.
FAU - Etcheverrigaray, Marina
AU  - Etcheverrigaray M
AD  - Laboratorio de Cultivos Celulares, Facultad de Bioquimica y Ciencias Biologicas, 
      Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 
      242, S3000ZAA Santa Fe, Argentina.
FAU - Kratje, Ricardo
AU  - Kratje R
AD  - Laboratorio de Cultivos Celulares, Facultad de Bioquimica y Ciencias Biologicas, 
      Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 
      242, S3000ZAA Santa Fe, Argentina.
FAU - Prieto, Claudio
AU  - Prieto C
AD  - Laboratorio de Cultivos Celulares, Facultad de Bioquimica y Ciencias Biologicas, 
      Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 
      242, S3000ZAA Santa Fe, Argentina.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20131122
PL  - United States
TA  - Protein Expr Purif
JT  - Protein expression and purification
JID - 9101496
RN  - 0 (Peptide Elongation Factor 1)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - 839MOZ74GK (F8 protein, human)
RN  - 9001-27-8 (Factor VIII)
SB  - IM
MH  - Animals
MH  - CHO Cells
MH  - Cricetinae
MH  - Cricetulus
MH  - Cytomegalovirus/genetics
MH  - Factor VIII/chemistry/genetics/*metabolism
MH  - Genetic Vectors/*genetics
MH  - Green Fluorescent Proteins/chemistry/genetics/metabolism
MH  - HEK293 Cells
MH  - HeLa Cells
MH  - Humans
MH  - Lentivirus/*genetics
MH  - Peptide Elongation Factor 1/genetics
MH  - Promoter Regions, Genetic/genetics
MH  - Protein Engineering
MH  - Recombinant Fusion Proteins/chemistry/genetics/*metabolism
OTO - NOTNLM
OT  - CMV enhancer/promoter
OT  - EF-1alpha promoter
OT  - Lentiviral vector
OT  - Protein overexpression
OT  - rhFVIII production
EDAT- 2013/11/28 06:00
MHDA- 2014/10/09 06:00
CRDT- 2013/11/27 06:00
PHST- 2013/08/19 00:00 [received]
PHST- 2013/10/17 00:00 [revised]
PHST- 2013/11/14 00:00 [accepted]
PHST- 2013/11/27 06:00 [entrez]
PHST- 2013/11/28 06:00 [pubmed]
PHST- 2014/10/09 06:00 [medline]
AID - S1046-5928(13)00242-8 [pii]
AID - 10.1016/j.pep.2013.11.005 [doi]
PST - ppublish
SO  - Protein Expr Purif. 2014 Mar;95:50-6. doi: 10.1016/j.pep.2013.11.005. Epub 2013 
      Nov 22.