PMID- 35205024
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20220228
IS  - 2079-7737 (Print)
IS  - 2079-7737 (Electronic)
IS  - 2079-7737 (Linking)
VI  - 11
IP  - 2
DP  - 2022 Jan 19
TI  - A De Novo Optimized Cell-Free System for the Expression of Soluble and Active 
      Human Tumor Necrosis Factor-Alpha.
LID - 10.3390/biology11020157 [doi]
LID - 157
AB  - Cell-free (in vitro) expression is a robust alternative platform to the 
      cell-based (in vivo) system for recombinant protein production. Tumor necrosis 
      factor-alpha (TNF-alpha) is an effective pro-inflammatory cytokine with pleiotropic 
      effects. The aim of the current study was de novo optimized expression of soluble 
      and active human TNF-alpha by an in vitro method in an E. coli-based cell-free 
      protein synthesis (CFPS) system and its biological activity evaluation. The 
      codon-optimized synthetic human TNF-alpha gene was constructed by a two-step PCR, 
      cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. 
      Cell-free expression of the soluble protein was optimized using a response 
      surface methodology (RSM). The anticancer activity of purified human TNF-alpha was 
      assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data 
      from RSM revealed that the lowest value (7.2 microg/mL) of cell-free production of 
      recombinant human TNF-alpha (rhTNF-alpha) was obtained at a certain incubation time (6 h) 
      and incubation temperature (20  degrees C), while the highest value (350 microg/mL) was 
      recorded at 4 h and 35  degrees C. This rhTNF-alpha showed a significant anticancer potency. 
      Our findings suggest a cell-free expression system as an alternative platform for 
      producing soluble and functionally active recombinant TNF-alpha for further research 
      and clinical trials.
FAU - El-Baky, Nawal Abd
AU  - El-Baky NA
AUID- ORCID: 0000-0002-1738-2274
AD  - Therapeutic and Protective Proteins Laboratory, Protein Research Department, 
      Genetic Engineering and Biotechnology Research Institute (GEBRI), City of 
      Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab 
      City, Alexandria P.O. Box 21934, Egypt.
FAU - El-Fakharany, Esmail M
AU  - El-Fakharany EM
AD  - Therapeutic and Protective Proteins Laboratory, Protein Research Department, 
      Genetic Engineering and Biotechnology Research Institute (GEBRI), City of 
      Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab 
      City, Alexandria P.O. Box 21934, Egypt.
FAU - Sabry, Soraya A
AU  - Sabry SA
AD  - Department of Botany and Microbiology, Faculty of Science, Alexandria University, 
      Alexandria P.O. Box 21568, Egypt.
FAU - El-Helow, Ehab R
AU  - El-Helow ER
AUID- ORCID: 0000-0002-4319-3856
AD  - Department of Botany and Microbiology, Faculty of Science, Alexandria University, 
      Alexandria P.O. Box 21568, Egypt.
FAU - Redwan, Elrashdy Mustafa
AU  - Redwan EM
AUID- ORCID: 0000-0001-8246-0075
AD  - Biological Sciences Department, Faculty of Science, King Abdulaziz University, 
      Jeddah P.O. Box 80203, Saudi Arabia.
FAU - Sabry, Amira
AU  - Sabry A
AD  - Therapeutic and Protective Proteins Laboratory, Protein Research Department, 
      Genetic Engineering and Biotechnology Research Institute (GEBRI), City of 
      Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab 
      City, Alexandria P.O. Box 21934, Egypt.
LA  - eng
PT  - Journal Article
DEP - 20220119
PL  - Switzerland
TA  - Biology (Basel)
JT  - Biology
JID - 101587988
PMC - PMC8868817
OTO - NOTNLM
OT  - anticancer
OT  - human tumor necrosis factor-alpha
OT  - in vitro protein synthesis
OT  - optimization
OT  - response surface methodology
COIS- The authors declare no conflict of interest.
EDAT- 2022/02/26 06:00
MHDA- 2022/02/26 06:01
PMCR- 2022/01/19
CRDT- 2022/02/25 01:05
PHST- 2021/11/09 00:00 [received]
PHST- 2022/01/14 00:00 [revised]
PHST- 2022/01/17 00:00 [accepted]
PHST- 2022/02/25 01:05 [entrez]
PHST- 2022/02/26 06:00 [pubmed]
PHST- 2022/02/26 06:01 [medline]
PHST- 2022/01/19 00:00 [pmc-release]
AID - biology11020157 [pii]
AID - biology-11-00157 [pii]
AID - 10.3390/biology11020157 [doi]
PST - epublish
SO  - Biology (Basel). 2022 Jan 19;11(2):157. doi: 10.3390/biology11020157.