Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin

Melina Laguia-Becher, Zurima Zaldúa, Weijie Xu, Patricia Laura Marconi, William Velander, María Alejandra Alvarez

Research output: Contribution to journalArticle

Abstract

The serine protease α-thrombin (FIIa) plays a fundamental role in blood clotting. In the present report, a FIIa precursor (pFIIa) was expressed in Nicotiana benthamiana Domin. The expression construct featured the Kozak consensus sequence and the 2S2 Arabidopsis thaliana (L.) Heynh. signal peptide to direct the protein into the secretory pathway (sec-pFIIa). A version carrying the KDEL endoplasmic reticulum (ER) retention signal (pFIIa-ER) was also constructed. Transient expression of pFIIa in N. benthamiana leaves was achieved by Agrobacterium tumefaciens infiltration. The influence of post-transcriptional gene silencing (PTGS) was analyzed by co-infiltrating with an A. tumefaciens strain carrying the construct for the Turnip Crinkle Virus-coat protein (TCV-CP) known for interfering with PTGS. Reverse transcription polymerase chain reaction and Western blot analyses confirmed the presence of the corresponding messenger RNA and the recombinant pFIIa protein in plant extracts. A positive effect of the addition of the PTGS inhibitor was demonstrated. The accumulation of sec-pFIIa and pFIIa-ER was estimated to be 6 μg g −1 fresh weight (FW) (0.07% (w/w) total protein concentration; TPC) and 17 μg g −1 FW (0.21% (w/w) TPC), respectively. Furthermore, stably transformed callus and suspension cultures were obtained. The recombinant protein was detected only in the biomass of the pFIIa-ER cell suspension line at a concentration of 0.25 μg mL −1 (0.017% (w/w) of total soluble protein). This appears to be the first report describing the expression of a precursor of FIIa in plants.

Original languageEnglish (US)
Pages (from-to)88-98
Number of pages11
JournalIn Vitro Cellular and Developmental Biology - Plant
Volume55
Issue number1
DOIs
StatePublished - Feb 15 2019

Fingerprint

Carmovirus
Turnip crinkle virus
thrombin
Nicotiana benthamiana
Capsid Proteins
Serine Proteases
serine proteinases
coat proteins
Thrombin
Endoplasmic Reticulum
endoplasmic reticulum
Tobacco
RNA Interference
RNA interference
Agrobacterium tumefaciens
Agrobacterium radiobacter
Recombinant Proteins
recombinant proteins
Suspensions
Weights and Measures

Keywords

  • Agrobacterium tumefaciens infiltration
  • Alpha-thrombin
  • Plant cell suspension cultures
  • Plant-made recombinant protein
  • Post-transcriptional gene silencing

ASJC Scopus subject areas

  • Biotechnology
  • Plant Science

Cite this

Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin. / Laguia-Becher, Melina; Zaldúa, Zurima; Xu, Weijie; Marconi, Patricia Laura; Velander, William; Alvarez, María Alejandra.

In: In Vitro Cellular and Developmental Biology - Plant, Vol. 55, No. 1, 15.02.2019, p. 88-98.

Research output: Contribution to journalArticle

Laguia-Becher, Melina ; Zaldúa, Zurima ; Xu, Weijie ; Marconi, Patricia Laura ; Velander, William ; Alvarez, María Alejandra. / Co-expressing Turnip Crinkle Virus-coat protein with the serine protease α-thrombin precursor (pFIIa) in Nicotiana benthamiana Domin. In: In Vitro Cellular and Developmental Biology - Plant. 2019 ; Vol. 55, No. 1. pp. 88-98.
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abstract = "The serine protease α-thrombin (FIIa) plays a fundamental role in blood clotting. In the present report, a FIIa precursor (pFIIa) was expressed in Nicotiana benthamiana Domin. The expression construct featured the Kozak consensus sequence and the 2S2 Arabidopsis thaliana (L.) Heynh. signal peptide to direct the protein into the secretory pathway (sec-pFIIa). A version carrying the KDEL endoplasmic reticulum (ER) retention signal (pFIIa-ER) was also constructed. Transient expression of pFIIa in N. benthamiana leaves was achieved by Agrobacterium tumefaciens infiltration. The influence of post-transcriptional gene silencing (PTGS) was analyzed by co-infiltrating with an A. tumefaciens strain carrying the construct for the Turnip Crinkle Virus-coat protein (TCV-CP) known for interfering with PTGS. Reverse transcription polymerase chain reaction and Western blot analyses confirmed the presence of the corresponding messenger RNA and the recombinant pFIIa protein in plant extracts. A positive effect of the addition of the PTGS inhibitor was demonstrated. The accumulation of sec-pFIIa and pFIIa-ER was estimated to be 6 μg g −1 fresh weight (FW) (0.07{\%} (w/w) total protein concentration; TPC) and 17 μg g −1 FW (0.21{\%} (w/w) TPC), respectively. Furthermore, stably transformed callus and suspension cultures were obtained. The recombinant protein was detected only in the biomass of the pFIIa-ER cell suspension line at a concentration of 0.25 μg mL −1 (0.017{\%} (w/w) of total soluble protein). This appears to be the first report describing the expression of a precursor of FIIa in plants.",
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