TY - JOUR

T1 - Tick-borne encephalitis nonstructural protein NS1 expressed in E. coli retains immunological properties of the native protein

AU - Andrey, Matveev

AU - Yana, Khlusevich

AU - Olga, Golota

AU - Bogdana, Kravchuk

AU - Sergey, Tkachev

AU - Lyudmila, Emelyanova

AU - Nina, Tikunova

N1 - Funding Information: The research was funded by RFBR and Novosibirsk region , project number 20-44-54301 3. Publisher Copyright: © 2021 Elsevier Inc.

PY - 2022/3

Y1 - 2022/3

N2 - There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

AB - There is evidence that flaviviral NS1 glycoprotein plays an important role in the pathology of tick-borne encephalitis (TBE) and NS1-specific antibodies are detected in the blood of patients with TBE. This makes NS1 a good target for the development of therapeutic inhibitors and NS1 could be an important biomarker for the early diagnosis of TBE in vaccinated individuals. Eukaryotic expression systems are mainly used to produce recombinant tick-borne encephalitis virus (TBEV) NS1. The expression of TBEV NS1 proteins in eukaryotic cells was successful, but there were some limitations. Several attempts have also been made to obtain the NS1 protein in Escherichia coli cells; however, they were unsuccessful due to the low solubility of the recombinant protein and improper folding. In this study, using Trx-tag as a fusion partner, soluble Trx-fused TBEV NS1 protein was first produced in the E. coli BL21 strain. In addition, insoluble Trx-fused TBEV NS1 protein was obtained when cultivation conditions were changed to increase the productivity. The insoluble TBEV NS1 obtained from inclusion bodies was solubilized using chaotropic reagents and successfully refolded using dialysis. Both soluble variant and successfully refolded from inclusion bodies variant showed immunological properties similar to the native TBEV NS1 protein and were recognized by specific monoclonal antibodies (mAbs), immune ascetic fluid in ELISA, western blot, and competitive analysis.

KW - E. coli expression

KW - NS1 protein

KW - Recombinant protein

KW - Tick-borne encephalitis

KW - Trx-fused protein

UR - http://www.scopus.com/inward/record.url?scp=85121117727&partnerID=8YFLogxK

UR - https://www.elibrary.ru/item.asp?id=47540705

U2 - 10.1016/j.pep.2021.106031

DO - 10.1016/j.pep.2021.106031

M3 - Article

C2 - 34920135

AN - SCOPUS:85121117727

VL - 191

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

M1 - 106031

ER -