CC BY
2DOI https://doi.org/10.47612/1999-9127-2021-30-105-109 УДК 579.66:577.217.3+579.842.11
I. S. Kazlouski1, I. V. Belskaya2, A. B. Bulatovskiy1, A. I. Zinchenko1
THE USE OF A CELL-FREE PROTEIN SYNTHESIS FOR OBTAINING BACTERIAL DIGUANYLATCYCLASE AND TWO CHIMERIC
PROTEINS
:State Scientific Institution "Institute of Microbiology of the National Academy of Sciences of Belarus" 2 Kuprevich St., 220141 Minsk, Republic of Belarus
e-mail: kazlouski.illia@gmail.com 2The Republican Research and Practical Center for Epidemiology and Microbiology 23 Filimonova St., 220114 Minsk, Republic of Belarus
The novel promising trend of biotechnology is cell-free synthesis of proteins. Possibility of producing two chimeric proteins: adenosine deaminase of Escherichia coli fused with human annexin-A5, DNA-affine domain of bacteria Sulfolobus solfataricus fused with modified Taq-DNA-polymerase and bacterial enzyme diguanyl-atecyclase of Thermotoga maritima, by cell-free synthesis procedure as an alternative to conventional cultivation of microbial strains-producers in a bioreactor was studied in this research. Chimeric RNA polymerase of T7 bacteriophage, S30-cell extract of E. coli and multicopy plasmid pET42mut were engaged for protein synthesis by cell-free protein synthesis system. The first successful production of these proteins was demonstrated in CFPS system.
Keywords: chimeric protein, diguanylatecyclase, annexin-A5, adenosine deaminase, SSo-Taq-polymerase, Esch-erichia coli, cell-free protein synthesis system, multicopy plasmid.
Introduction
Currently, biotechnology based on genetic engineering is widely used to obtain drugs of peptide or protein nature [1]. Despite notable success, this approach has significant limitations:
1) not all genes are expressed in a "foreign environment";
2) the expression of polycistronic genes has not been demonstrated so far;
3) post-translational modification and formation of the structure of multidimensional proteins of eukaryotes can not be realized in bacterial cells.
A number of research groups use cell-free systems as an alternative to the conventional whole-cell technology to produce valuable proteins, especially membranes, therapeutic proteins and polypeptides containing unnatural amino acids [2].
The system of cell-free protein synthesis (CFPS) provides for gene transcription and translation of mRNA in vitro in a cell lysate. The ly-sate contains recombinant DNA, amino acids,
nucleotides, cofactors, and ATP-regenerating system. However, endogenous genetic information (DNA and mRNA) is removed.
Compared to the whole cells systems, CFPS systems offer several options. Noteworthy is production of exceptionally target protein, the opportunity to synthesize proteins toxic for cells; the possibility to synthesize proteins that contain unnatural amino acids, and as the main advantage, it becomes possible to resolve the problem of aggregation by adding into reaction mixture the agents maintaining the synthesized polypeptide in soluble form.
The main goal of this study was production of several recombinant proteins in the CFPS system. Their synthesis was problematic by the traditional methods relying on whole bacterial cells. In particular, it was planned to synthesize Thermotoga maritima diguanylatecyclase (DGC). The enzyme condenses GTP into a promising new generation adjuvant as cyclic diguanyl monophosphate [3]. In addition, we were interested in two chimeric proteins: the
pharmacologically valuable human annexin-A5 fused with Escherichia coli adenosine deaminase (Annexin-ADase) [4], and DNA polymerase with improved properties due to attachment to Taq-DNA polymerase DNA-binding domain (SSo7D) of the bacterium Sulfolobus solfatari-cus (Sso-Taq polymerase) [5].
Materials and Methods
DNA from T. maritima, low-copy plasmids pET42-A5-add [4] and pET18b-STaq [5] served as sources of genes encoding DGC (TM1788), Annexin-Adase (anxA5-add), and Sso-Taq-polymerase (SSO_RS123 75-taq) respectively. The target DNA fragments were amplified by polymerase chain reaction (PCR) using synthetic primers. The selection of primers was carried out using the UGENE 1.22 software (UniPro, Russia) based on the nucleotide sequences of the target genes. Sequences complementary to plasmid pET42mut were added to the 5'-ends of the primers [6]. Amplification was carried out according to the following program: pre-denaturation stage (30 s at 98 °C) — 30 amplification cycles (10 s at 98 °C; 15 s at 55 °C; 1 min at 72 °C) — final elongation 75 s at 72 °C. At the second stage, the pET42mut plasmid was linearized by the method described earlier [6].
During the next stage, the linearized vector and target genes were recovered by overlap extension PCR (OV-PCR) [7] according to the following protocol: Initial denaturation stage (30 s at 98 °C) — 16 amplification cycles (10 s at 98 °C; 15 s at 50 °C ; 4 min at 72 °C) — Final extension for 5 min at 72 °C. At this stage, the fragments resulting from the first two stages were used in equimolar amounts as a template and a primer.
The product synthesized from OV-PCR was applied to transform competent cells of E. coli XL1 Blue (Novagen, USA) by electroporation technique, followed by inoculation on a solid nutrient medium with kanamycin (100 p,g / ml). The cells with integrated plasmids were cultured in the liquid LB medium until subsequent extraction of plasmid DNA by alkaline hydrolysis.
The protein synthesis was performed in 1.0 ml of the reaction mixture containing 0.25 ml of the S30 cell extract E. coli, 0.65 ml of the
premix, 5000 units Sso7d-RNA polymerase of bacteriophage T7 [8], 500 ng of plasmid DNA obtained at the previous stage. The reaction mixture was incubated at 30 °C for 5-6 h. The activity of the target enzymes was determined according to the methods described in [3], [4] and [5].
Results and Discussion
The first research stage was focused on the amplification of the TM1788, anxA5-add, and SSO_RS123 75-taq genes encoding the amino acid sequences of DGC, Annexin-ADase, and Sso-Taq polymerase, respectively.
Horizontal DNA electrophoresis in agarose gel after isolation and amplification of genes proved that nucleotide sequences of the required sizes were obtained: TM1788 — 750 bp, anxA5-add — 2000 bp. and SSORS12375-taq — 2800 bp. (fig. 1).
Fig. 1. Electropherogram of amplification products
of genes TM1788 (1), anxA5-add (2) and SSO_ RS12375-taq (3); M — molecular weight marker of DNA fragments
At the next stage, the pET42mut vector was linearized using PCR, and then OV-PCR was performed. In contrast to standard PCR, it requires the presence of overlapping complementary regions in the vector and insertions added at the stage of cloning the target genes. Thus,
the genes of interest TM1788, anxA5-add and SSO_RS12375-taq were inserted into the plas-mid pET42mut.
Competent E. coli XL1 Blue cells were transformed with polynucleotide material originating OV-PCR. Plasmids were isolated from the grown transformation cells, which were further subjected to sequencing to confirm the lack of unwanted spontaneous mutations.
At the next stage, the efficiency of DGC, Annexin-ADase, and SSo-Taq-polymerase synthesis in the CFPS system was evaluated. The final concentration of the reaction components was 100 mM HEPES-KOH (pH 8.0), 8 mM magnesium acetate, 90 mM potassium acetate, 20 mM potassium phosphoenolpyru-vate, a set of amino acids (each at 1.3 mM concentration), 0.15 mg / ml of folic acid, each of four ribonucleoside-5'-triphosphates at 1 mM concentration, 0.05% sodium azide, 2% polyethylene glycol-8000, 0.04 mg / ml pyruvate kinase, 5.5 p,g / ml Sso7d-RNA polymerase phage T7, 0.3 mg / ml plasmid DNA-contain-ing genes encoding the target proteins, 0.5 mg / ml total tRNA and S30 extract obtained from E. coli cell lysate (30% of the total vol-
ume of the reaction mixture). The incubation lasted for 6 h at 30 °C with moderate stirring. 1 sample was taken every hour for analysis of the enzymatic activity of the synthesized proteins in order to control the synthesis of enzymes. A reaction mixture containing appropriate amount of buffer solution was used as a negative control instead of plasmid DNA. The results of the synthesis of Sso-Taq-poly-merase, Annexin-ADase, and DGC are shown in figure 2.
The final concentrations of DGC, Annexin-ADase, and Sso-Taq polymerase were 45 U / ml, 5 U / ml, and 250 U / ml respectively in the resulting enzyme preparations. The highest concentrations of these proteins were 0.0036 U / ml [3], 0.1325 U / ml [4] and 240 U / ml [5], respectively, in the cultural liquids, following their production in whole-cell expression systems. It can be seen that the expression level of SSo-Taq polymerase is close to the whole cell level, which may indicate that the conditions for the synthesis of this enzyme in the CFPS system were not fully optimized and it is essential to continue the studies of this process.
Fig. 2. Dynamics of the accumulation of recombinant proteins in the reaction mixture during synthesis in the CFPS
system
Conclusion
The first successful production of bacterial enzyme DGC T. maritima and two chimeric proteins (Annexin-ADase and Sso-Taq polymerase) was demonstrated in CFPS system.
For the first time, the CFPS system was used to obtain the bacterial enzyme DGC T. maritima, and two chimeric proteins (Annexin-ADase and Sso-Taq polymerase).
After partial optimization of the reaction conditions, 1 ml samples of DGC, Annexin-ADase and Sso-Taq-polymerase enzyme preparations with activities 45 U / ml, 5 U / ml and 250 U / ml, respectively, were obtained. In our opinion, the synthesis of these proteins in the CFPS system can be an alternative to submerged cultivation of microbial strains-producers in a bioreactor.
References
1. Sambyal, K. Bioprocess and genetic engineering aspects of ascomycin production: a review / K. Sambyal, R. V. Singh // J. Genet. Eng. Biotechnol. - 2020. - Vol. 18. - P. 73.
2. Cell-free protein synthesis: a promising option for future drug development / S. K. Dondapati [et al.] // BioDrugs. - 2020. - Vol. 34. - P. 327348.
3. Enzymatic synthesis of c-di-GMP using inclusion bodies of Thermotoga maritima full-length diguanylate cyclase / A. S. Korovashkina
[et al.] // J. Biotechnol. - 2012. - Vol. 164. -P. 2276-2280.
4. Construction of strain-producer of chemeric protein containing human annexin and bacterial adenosine deaminase / A. B. Bulatovski [et al.] // Doklady of the National Academy of Sciences of Belarus. - 2017. - Vol. 61, № 4. - P. 89-95.
5. Production of thermostable DNA polymerase suitable for whole-blood polymerase chain reaction / A. S. Korovashkina [et al.] // Biochemistry and Biotechnology: Research and Development / Eds: S. D. Varfolomeev, G. E. Zaikov, L. P. Kry-lova. New York, Nova Science Publishers, Inc. -2012. - P. 1-5.
6. Kazlouski, I. S. Modifikaciya ekspressionnoj pET-sistemy dlya ispolzovaniya v beskletochnom sinteze belka / I. S. Kazlouski, A. N. Rymko, A. I. Zinchenko // Sb. nauch. trudov Instituta mi-krobiologii NAN Belarusi «Mikrobnie biotekh-nologii: fundamentalnie i prikladnie aspekti». -2018. -Vol. 10. - P. 69-78.
7. Quan, J. Circular polymerase extension cloning of complex gene libraries and pathways / J. Quan, J. Tian // PloS ONE. - 2009. - Vol. 4, № 7. -P. 6441-6442.
8. Kazlouski, I. S. Sozdanie shtamma-produ-centa himernogo belka, sostoyashchego iz RNK-polimerazi i DNK-affinnogo domena / I. S. Kazlouski, A. I. Zinchenko // Dokl. Nac. akad. nauk Belarusi. - 2018. - Vol. 62, № 5. - P. 601-607.
И. С. Казловский1, И. В. Бельская2, А. Б. Булатовский1, А. И. Зинченко1
ИСПОЛЬЗОВАНИЕ СИСТЕМЫ БЕСКЛЕТОЧНОГО СИНТЕЗА БЕЛКА ДЛЯ ПОЛУЧЕНИЯ БАКТЕРИАЛЬНОЙ ДИГУАНИЛАТЦИКЛАЗЫ И ДВУХ ХИМЕРНЫХ БЕЛКОВ
Государственное научное учреждение «Институт микробиологии Национальной академии наук Беларуси» Республика Беларусь, 220141, г. Минск, ул. Купревича, 2 e-mail: kazlouski.illia@gmail.com Республиканский научно-практический центр эпидемиологии и микробиологии Республика Беларусь, 220114, г. Минск, ул. Филимонова, 23
Новым перспективным направлением в биотехнологии является бесклеточный синтез белка (БСБ). В представленном исследовании изучена возможность синтеза в бактериальной системе БСБ дигуанилатцикла-зы (ДГЦ) бактерии Thermotoga maritima и двух химерных белков: человеческого аннексина-А5, слитого с аденозиндезаминазой Escherichia coli (аннексин-АДаза), и модифицированной Taq-ДНК-полимеразы, слитой с ДНК-аффинным доменом бактерии Sulfolobus solfataricus (SSo-Taq-полимераза). В реакционной смеси для бесклеточного синтеза этих белков использовали химерную РНК-полимеразу бактериофага Т7, клеточный экстракт E. coli, и высококопийную плазмиду pET42mut. В результате исследования впервые показана возможность синтеза вышеупомянутых белков в системе БСБ.
Ключевые слова: химерный белок, дигуанилатциклаза, аннексин-А5, аденозиндезаминаза, SSo-Taq-полимераза, Escherichia coli, бесклеточный синтез белка, высококопийная плазмида.
Дата поступления статьи: 31 марта 2021 г.
