Artículo Original

 

Expression of recombinant PreS2-S protein from the hepatitis B virus surface antigen in Pichia pastoris

Expresión de la proteína recombinante PreS2-S del antígeno de superficie del virus de la hepatitis B en Pichia pastoris

 

Sobirdjan A. Sasmakov*   ORCID: https://orcid.org/0000-0003-1863-7117

Oybek N. Ashirov   ORCID: http://orcid.org/0000-0002-0855-6105

Jaloliddin M. Abdurakhmanov   ORCID: https://orcid.org/0000-0002-3954-3251

Shukhrat Sh. Khasanov   ORCID: https://orcid.org/0000-0003-4773-7117

Farkhod B. Eshboev   ORCID: https://orcid.org/0000-0003-3054-6111

Elvira G. Yusupova   ORCID: https://orcid.org/0000-0003-4100-0632

Galina A. Piyakina   ORCID: https://orcid.org/0000-0001-5370-9900

Shakhnoz S. Azimova   ORCID: https://orcid.org/0000-0003-1803-7728

Acad. S. Yu. Yunusov Institute of the Chemistry of Plant Substances, M. Ulugbek Str. 77, 100170, Tashkent, Uzbekistan.

Autor para correspondencia: sasmakov@web.de

 

ABSTRACT

The aim of this work is the expression of the PreS2-S region of surface antigen of hepatitis B virus (HBV) in yeast Pichia pastoris. A cDNA fragment encoding the Pres2-S protein of HBV was cloned to yeast transfer vectors. Based on cloned new plasmids pPIC3.5-PreS2-S (8707 bp) and pPIC9-PreS2-S (8980 bp) the recombinant strains of P. pastoris producing the PreS2-S region of surface antigen of HBV were obtained. The PAGE electrophoresis and immunoblotting of obtained recombinant PreS2-S protein confirm the molecular weight (34 kDa) and high specificity to the HBV antibodies.

Keywords: Pichia; hepatitis B virus; recombinant DNA; recombinant proteins.

 

RESUMEN

El objetivo de este trabajo es la expresión de la región PreS2-S del antígeno de superficie del virus de la hepatitis B en la levadura Pichia pastoris. Se clonó un fragmento de ADNc que codifica la proteína PreS2-S del VHB en vectores de transferencia de levadura. A partir de los nuevos plásmidos clonados pPIC3.5-PreS2-S (8707 pb) y pPIC9-PreS2-S (8980 pb) se obtuvieron las cepas recombinantes de P. pastoris productoras de la región PreS2-S del antígeno de superficie del VHB. La electroforesis PAGE y la inmunotransferencia de la proteína PreS2-S recombinante obtenida confirman el peso molecular (34 kDa) y la alta especificidad a los anticuerpos contra el VHB.

Palabras clave: Pichia; virus de la hepatitis B; ADN recombinante; proteínas recombinantes.

 

 

Submitted: June 25, 2020

Approved: September 24, 2020

 

 

Introduction

Nowadays, 292 million people are chronically infected with hepatitis B virus (HBV), as a result, 1.4 million of people die each year in the world.(1,2) This infectious disease is in second place after tuberculosis in terms of mortality and the number of infected people with hepatitis is higher than the number of infected people with HIV. However, over 80% of people infected with hepatitis do not have access to prevention, testing, and treatment.(3)

Currently, the HBV virus vaccine, which is a recombinant hepatitis B surface antigen protein based on the HBV-S region, is used to the prevention of this disease in the world. This protein (consists of 226 amino acids) contains only one immunogenic determinant "a". It is common to all genotypes of HBV and cannot provide complete protection against the virus.(4)

It is known that proteins encoded by PreS1 (108 a.a.), PreS2 (55 a.a.), and S (226 a.o.) regions of the virus genome are considered highly immunogenic and a part of its outer surface. It is considered that the PreS2-S (PreS2 + S) region of the HBV surface protein (containing 281 amino acids) has polyalbumin-binding activity, which indicates the complementarity of hepatocyte surface receptors.

Recent studies have shown that the presence of PreS2-S protein in the hepatitis B virus vaccine contributes to a significant increase in the immunogenicity of the vaccine.(5,6,7,8,9) Today, various expression systems are used to obtain this protein, including yeast expression system.(10,11)

Pichia pastoris methylotrophic yeast is one of the widely used microorganisms for the production of heterologous proteins. The advantages of the P. pastoris expression system are: the accumulation of significant biomass during cultivation on inexpensive nutrient media, the absence of endotoxins and pyrogens, a higher level of synthesis of recombinant proteins and the ability to synthesize recombinant protein in a nutrient medium.(12,13,14,15)

Various vectors are currently available for use in this expression system. In the present work, the transfer vectors pPIC3.5 (7751 b.p) and pPIC9 (8023 b.p) containing the nucleotide sequences of the P. pastoris genome, including the AOX1 promoter, HIS4 were used in order to obtain new recombinant plasmids encoding the PreS2-S region of the HBV of genotype D commonly widespread in Uzbekistan.(16)

 

Materials and Methods

In the experiments, enzymes and reagents from Thermo Fisher Scientific (USA), New England Biolabs (USA), Sigma-Aldrich (Merck, Germany), SBS Genetech (China), Panreac (Germany), Biosset (Russia), Himedia (India) were used.

 

Preparation of vectors and insertion (gene) for ligation

A bacterial strain of Escherichia coli containing the transfer vectors pPIC3.5 and pPIC9 (Invitrogen, Cat. no. K1710-01) was cultivated on Luria Bertani medium in the presence of ampicillin (30 μg/mL) at a temperature of 37°C.(17,18)

One microgram each of vectors pPIC3.5 and pPIC9 was digested sequentially with BamH I/EcoR I and Xho I/EcoR I, respectively.(18) Linear DNA molecules with length of 7745 bp and 7999 bp were isolated after electrophoresis in a 0.7% agarose gel by the method of electroelution.

The PreS2-S gene was prepared from the plasmid pBacPAK-8-Polh-PreS2-S by digestion with restriction enzymes BamHI/EcoRI for insertion into the transfer vector pPIC3.5 and XhoI/EcoRI for pPIC9.(16,19) The nucleotide sequence of Pres2-S gene fragment can be found in ENA with accession number LR745788.(20)

 

Ligation and transformation of the obtained plasmids into electrocompetent cells of E. coli Neb-5α. Analysis of the obtained clones

Ligation was performed using a T4 phage DNA-ligase with a prepared vector into a molar ratio of 1:10 (vector: insert) according to the standard method.(17) Then, the ligase mixture was transformed into competent cells of the E. coli NEB-5α.

The size of DNA plasmid isolated from clones that were obtained after transformation with a ligase mixture was determined by the method of agarose gel electrophoresis. Clones containing DNA with a molecular weight of 8.707 and 8.980 kb were investigated by PCR analysis using the following specific primers:

For pPIC3.5-PreS2-S

1. 5`-CGGATCCAAAAATGTCTCAGTGGAAC-3`

2. 5`-TGTTGAATTCAATGTATACCCAAA-3`

For pPIC9-PreS2-S

1. 5`-CCTCGAGAAAAATGTCTCAGTGGAAC-3`

2. 5`-TGTTGAATTCAATGTATACCCAAA-3`

After that, the products of amplification were analyzed by electrophoresis in a 1% agarose gel. DNA of the recombinant plasmids identified by PCR was also confirmed with restriction analysis. For this, a comparative analysis of the recombinant clones and the initial vector plasmids pPIC3,5 and pPIC9 were restricted by the EcoRI site.(18) The molecular size of the obtained fragments was determined by electrophoresis in a 0.7% agarose gel; 1 kb Plus DNA Ladder from Invitrogen (Cat. No 10787018) was used as a molecular weight marker.

 

Transformation of the obtained recombinant plasmids to the P. pastoris yeast strain and identification of the obtained clones

The transformation of the obtained recombinant plasmids containing the target genes to the P. pastoris was conducted by the method of electroporation.(18) Integration of the target gene into the genome of GS115 P. pastoris strain was detected by PCR using the following primers:

5′ AOX1 -5′-GACTGGTTCCAATTGACAAGC-3′;

3′ AOX1 -5′-GCAAATGGCATTCTGACATCC-3′;

α-factor -5′-TACTATTGCCAGCATTGCTGC -3′.

 

ELISA and Western blotting analysis of recombinant PreS2-S protein

ELISA was performed according to Clark and Adams.(21) The recombinant PreS2-S protein was determined using test systems of the “ORTO Diagnostics” (USA) and “Diagnostic test systems of ICPS AS RUz” (Uzbekistan). The recombinant protein was detected by the corresponding antibody conjugated with alkaline phosphate diluted in conjugate buffer (PBS-TPO, pH 7.4) according to the suppliers’ specifications. Absorbance values were measured at 450 nm with a microplate reader (ELx800 Universal Microplate Reader, Bio-Tek Instruments Belgium). A sample was considered to be positive if its absorbance value was greater at least two times than absorbance values of the negative control. Whole cell-lysate of non-containing PreS2-S gene of GS115 P. pastoris cells was used as a negative control. For the analysis of Western blotting was used a commercial kit “Western Breeze, Chromogenic Western Blot Immunodetection Kit” (Invitrogen, Cat. WB7105).

 

Results and Discussion

According to their physical maps (Fig. 1), plasmids pPIC3.5 (A) and pPIC9 (B) were processed sequentially with restriction enzymes BamH I/EcoR I and Xho I/EcoR I in order to clone new inserts. The ligation of foreign DNA encoding the amino acid sequence of the protein into these sites, places the heterologous gene under the control of the promoter of the AOX1 gene.(18)

BamHI:

5′-G↓GATCC-3′ (pPIC3.5)

3′-CСTAG↓G-5′

XhoI:

5′-C↓TCGAG (pPIC9)

3′-GAGCT↓С-5′

EcoRI:

5′-G↓AATTC-3′ (pPIC3.5 and pPIC9)

3′-CTTAA↓G-5′

As the results, fragments of the vectors pPIC3.5 and pPIC9 with “sticky” ends that allowing for “directed” ligation were obtained. Linear DNA plasmids with a length of 7745 bp and 7999 bp were isolated after agarose gel electrophoresis. They were processed with alkaline phosphatase for avoid the "closure" of the vectors.

 

Fig. 1. Physical maps of transfer vectors pPIC3.5 (A) and pPIC9 (B)

 

The PreS2-S gene was isolated from plasmid pBacPAK-8-Polh-PreS2-S by digestion with appropriate restriction enzymes BamHI/EcoRI and XhoI/EcoRI.(16,19)

The digested transfer vectors were ligated to the fragment containing PreS2-S cDNA of the HBV region in a molar ratio of 1:10 by using T4 phage DNA ligase. After that, the obtained ligase mixture was transformed into the competent cells of E. coli NEB-5a. The identification of the formed clones was performed by PCR using the primers for the PreS2-S region. Then, PCR products were analyzed by electrophoresis in 1% agarose gel (Fig. 2).

Fig. 2. Gel electrophoresis of transformants containing the target gene.

A - pPIC3.5-PreS2-S; B - pPIC9-PreS2-S; C (K-) - DNA isolated from the blood of HBV negative people; D - DNA line (Marker); E (K +) - DNA isolated from the blood of HBV "positive" patients.

 

The presence of a DNA fragment (A and B) with a molecular size (956 bp) corresponding to the mass of the DNA fragment amplified from the HBV positive sample (E) indicated the presence of an insert in the studied plasmids. Thus, clones containing the PreS2-S region of HBV DNA were detected by PCR. Comparative restriction analysis of recombinant plasmids pPIC3.5-PreS2-S and pPIC9-PreS2-S with the original vectors pPIC3.5, pPIC9 by the EcoR I single site indicated that the molecular weight of recombinant plasmids corresponds to theoretical calculations on physical maps of these plasmids by the results of electrophoresis in a 0.7% agarose gel (Fig. 3). The linear forms of the recombinant plasmids pPIC3.5-Pres2-S and pPIC9-Pres2-S corresponding to 8707 bp and 8980 bp by the digestion with the EcoR I restriction enzyme were obtained.

Fig. 3. Restriction analysis of pPIC3.5-PreS2-S (EcoR I/8707 bp).

1- pPIC3.5/EcoR I (7751 bp); 2- pPIC3.5-PreS2-S/EcoR I (8707 bp)

 

Then, the transformation of recombinant plasmids pPIC3.5-PreS2-S and pPIC9- PreS2-S linearized in the HIS4 region by Sal I nuclease into the strain of P. pastoris yeast was conducted. Transformants (His +) were selected in histidine-deficient media. Obtained recombinant clones and integration of the target gene into the P. pastoris genome were identified by PCR using the α-factor primer (for pPIC9 only) and the 5´ AOX1 primer paired with the 3´ AOX1 primer.(18)

Several selected colonies of transformants were cultivated to the stage of the exponential phase (OD=2 at 600 nm) in a medium containing glycerin. The expression was induced by the addition of methanol until a final concentration of 0.5%, over 120 h of cultivation. The level of synthesis of recombinant protein and specificity were determined by the method of solid-phase enzyme linked immunoassay analyses (ELISA). For this purpose the samples (whole-cell lysates) were taken serially at 24 h intervals. Hepatitis-B Vaccine (rDNA) 20 µg/mL, Serum Institute of India and Euvax-B (Hepatitis B Vaccine [rDNA]), Sanofi Pasteur Korea Ltd., were used as the calibration standard (Table 1).

 

Table 1. Determination of recombinant Pres2-S protein level in whole-cell lysates of P. pastoris by ELISA depending on cultivation time.

The cultivation time after induction with methanol, hours

Result of ELISA         (OD value at 450 nm)

The amount of PreS2-S protein in the test sample

24

0.612

≈ 5 ng/mL

48

1.100

≈ 10 ng/mL

72

1.998

≈ 20 ng/mL

96

>3.000

≥ 26.5 ng/mL

120

2.890

≈ 25 ng/mL

Euvax-B (Hepatitis B Vaccine [rDNA]) 20 µg/mL  diluted:

1:4,000

0.532

≈ 5 ng/mL

1:2,000

0.971

≈ 10 ng/mL

1:1,000

2.178

≈ 20 ng/mL

1:750

>3.000

≥ 26.5 ng/mL

 

According to the results of ELISA, the optimal cultivation time was 96 h after induction with methanol.

The results of electrophoresis in PAGE and immunoblotting showed correspondence of the obtained recombinant protein to the expected molecular weight (about 34 kDa) and high specificity to the HBV antibodies (Fig. 4).

Fig. 4. The result of the immunoblot of the recombinant protein PreS2-S.

 

 

Conclusion

New recombinant plasmids pPIC3.5- PreS2-S and pPIC9- PreS2-S containing the cDNA PreS2-S region of the HBV surface antigen were cloned and recombinant strains of P. pastoris producing the target protein were obtained on their basis. The study of immunogenicity (humoral and cellular immunity) is required and may provide conclusive evidence of that the PreS2-S protein is an ideal candidate for a new generation of hepatitis B vaccines.

 

Conflict of interest

The Authors declare that there is no conflict of interest associated with this paper.

 

Author contributions

S.A. Sasmakov designed the study, interpreted the obtained results and provided the literature review.

O.N. Ashirov, J.M. Abdurakhmanov, Sh.Sh. Khasanov and F.B. Eshboev performed the experiments part of cloning of recombinant plasmids pPIC3.5-PreS2-S and pPIC9-PreS2-S.

E.G. Yusupova and G.A. Piyakina performed ELISA studies of recombinant Pres2-S protein and cultivated of experimental strains of P. pastoris.

Sh.S. Azimova participated in discussion of the obtained results and in writing the first draft of the manuscript.

All authors reviewed and approved the final version of this manuscript for publication.

 

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* PhD in Bioorganic Chemistry, Senior Researcher, Project Leader, Department of Molecular genetics.