New improved mammalian expression system

Shahrul Hisham Zainal Ariffin, Nurul Atikah Ahmad, Zafirah Azween Zainal Alam, Intan Zarina Zainol Abidin, Sahidan Senafi, Zulkiflie Zamrod, Rohaya Megat Abdul Wahab, Nurul Yuziana Mohd Yusof

Research output: Contribution to journalArticle

Abstract

Background and Objective: Mammalian cells are often used as a host for recombinant protein production although there are some other host such as yeast and bacteria. Nevertheless, more efficient mammalian expression system is needed due to the lack of potential mammalian expression system in producing recombinant protein. Hence, this study evaluates the efficiency of expression vectors that contain Matrix Attachment Region (MAR) and Integration Element (IE). Methodology: The MAR and IE sequences were identified using online database; MARFinder, MARScan, SMARTest and NCBI. Transfection on Chinese Hamster Ovary (CHO) cell was carried out using lipofectamine® LTX reagent. Expression analysis for transient transfection was carried out 24 h after transfection while expression analysis for stable transfection was performed from passage 1-4. Expression analysis was done using confocal fluorescence microscope and spectrofluorometer. Results: Bioinformatics analysis of these sequences resulted in selecting MAR_7_1_956 and H-1C. The new construct vectors are named pZAAM956 (MAR) and pZAAH1C (IE) and the vector without MAR and IE; pZAAGFP and commercial vector (phrGFP) were act as controls. All expression vectors carried Green Flouresent Protein (GFP) that emit green fluoresence light. Transfection analysis using microscopy approach shows the number of transfected cells for pZAAGFP, pZAH1C and pZAAM956 is more than 80% and there is significant difference (p<0.05) between pZAAH1C and phrGFP at stable transfection where the percentage of transfected cells of pZAAH1C is higher than phrGFP. This value is similar with the number of transfected cells for phrGFP. The GFP intensity of pZAAM956 showed the highest intensity measured by spectrofluorometer at emission wavelength 506 nm and excitation 500 nm at cell number 1×105 cell mL-1. Paired t-test showed significant difference (p<0.05) between pZAAM956 and phrGFP where the GFP intensity of pZAAM956 is higher than phrGFP. Expression analysis of stable transfection shows that the intensity of GFP produced by CHO cells which transfected with pZAAH1C is more stable with low decreasing intensity of GFP when compared to other expression vectors. Conclusion: These three constructed expression vectors were functioning; where pZAAM956 shows the highest GFP intensity while pZAAH1C is the most stable.

Original languageEnglish
Pages (from-to)178-187
Number of pages10
JournalJournal of Biological Sciences
Volume16
Issue number5
DOIs
Publication statusPublished - 2016

Fingerprint

Matrix Attachment Regions
Transfection
Proteins
Cell Count
Cricetulus
Recombinant Proteins
Ovary
Methyl Green
Computational Biology
Sequence Analysis
Microscopy
Yeasts
Fluorescence
Databases
Bacteria

Keywords

  • Integration element
  • Mammalian expression system
  • Matrix attachment regions element
  • Vector construction

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Medicine

Cite this

New improved mammalian expression system. / Zainal Ariffin, Shahrul Hisham; Ahmad, Nurul Atikah; Alam, Zafirah Azween Zainal; Abidin, Intan Zarina Zainol; Senafi, Sahidan; Zamrod, Zulkiflie; Megat Abdul Wahab, Rohaya; Mohd Yusof, Nurul Yuziana.

In: Journal of Biological Sciences, Vol. 16, No. 5, 2016, p. 178-187.

Research output: Contribution to journalArticle

Zainal Ariffin, Shahrul Hisham ; Ahmad, Nurul Atikah ; Alam, Zafirah Azween Zainal ; Abidin, Intan Zarina Zainol ; Senafi, Sahidan ; Zamrod, Zulkiflie ; Megat Abdul Wahab, Rohaya ; Mohd Yusof, Nurul Yuziana. / New improved mammalian expression system. In: Journal of Biological Sciences. 2016 ; Vol. 16, No. 5. pp. 178-187.
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AU - Ahmad, Nurul Atikah

AU - Alam, Zafirah Azween Zainal

AU - Abidin, Intan Zarina Zainol

AU - Senafi, Sahidan

AU - Zamrod, Zulkiflie

AU - Megat Abdul Wahab, Rohaya

AU - Mohd Yusof, Nurul Yuziana

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