• Journal of Microbiology and Biotechnology
• /
• 제29권2호
• /
• pp.304-310
• /
• 2019

Interleukin-21 is a common ${\gamma}$-chain cytokine that controls the immune responses of B cells, T cells, and natural killer cells. Targeting IL-21 to strengthen the immune system is promising for the development of vaccines as well as anti-infection and anti-tumor therapies. However, the practical application of IL-21 is limited by the high production cost. In this study, we improved IL-21 production by codon optimization and selection of appropriate signal peptide in CHO-K1 cells. Codon-optimized or non-optimized human IL-21 was stably transfected into CHO-K1 cells. IL-21 expression was 10-fold higher for codon-optimized than non-optimized IL-21. We fused five different signal peptides to codon-optimized mature IL-21 and evaluated their effect on IL-21 production. The best result (a 3-fold increase) was obtained using a signal peptide derived from human azurocidin. Furthermore, codon-optimized IL-21 containing the azurocidin signal peptide promoted $IFN-{\gamma}$ secretion and STAT3 phosphorylation in NK-92 cells similar to codon-optimized IL-21 containing original signal peptide. Collectively, these results indicate that codon optimization and azurocidin signal peptides provide an efficient approach for the high-level production of IL-21 as a biopharmaceutical.

• 한국미생물·생명공학회지
• /
• 제47권3호
• /
• pp.449-458
• /
• 2019

Hepatitis B treatments using immune therapy are gaining interest because of the improvements in dendritic cell performance for antigen presentation, which induces an appropriate immune response and raises patient survival rates. This research aims to produce a significant amount of the HBcAg antigen, which can induce an immune response and have a curative effect on HBV infection. In this study, the HBV subgenotype B3 of the HBcAg gene was used, which is dominant in Indonesia. Further, Lactococcus lactis bacteria was used as the host because of its safety and tightly regulated protein expression. The codon usage for the HBcAg gene was optimized to improve protein expression in L. lactis, which is important because a codon is not random between species. The HBcAg gene is attached to a pNZ8148 plasmid and transformed into the L. lactis NZ3900 expression host. The results confirm that a positive protein band (21 kDa) in two fractions of purified HBcAg was recognized by both western blotting and dot blot hybridization, even if the HBcAg optimized codon has higher GC contents than that suggested for L. lactis expression. Overall, this research strengthens the broad use of L. lactis bacteria for any protein expression, including higher protein expression of codon optimized HBcAg gene compared to non-optimized genes. Furthermore, the improvement in the codon optimization of the HBcAg gene significantly increases the total protein expression by 10-20%, and the expression level of the codon optimized HBcAg increases 1.5 to 3.2-times that of the native HBcAg.

• 한국산학기술학회논문지
• /
• 제21권9호
• /
• pp.559-568
• /
• 2020

세포 분열 및 성장 촉진에 영향을 주는 상피세포 성장인자(Epidermal Growth Factor, EGF)는 다양한 의학적 용도를 갖고 있는 호르몬 단백질이다. 본 연구에서는 human EGF 유전자를 대장균 코돈에 최적화 하고 pRSET 벡터에 클로닝하여 발현벡터를 구축하였다. Human EGF를 봉입체가 아닌 활성이 있는 형태로의 과량 발현을 위해 코돈의 최적화와 더불어 최초로 샤페론 공발현이 시도되었다. 발현된 Native protein 형태의 재조합 human EGF는 고순도로 정제하기 위해 Ion Exchange Chromatography를 2번 연속적으로 수행하여 순수 분리 정제되었고, ELISA 분석결과 99% 이상으로 재조합 EGF의 활성도가 상업용 EGF와 유사하게 나타났으며, 세포증식시험 결과 인간 재조합 EGF는 인체 피부 섬유아세포의 세포증식을 촉진하는 것으로 확인 되었다. 본 연구의 인간 EGF 발현 시스템은 양적인 측면 뿐 아니라 성공적인 활성형태의 발현으로 추가적인 재접힘 과정 및 N 말단의 융합부분을 제거하기 위한 크로마토그래피 작업이 필요가 없다는 점에서 기존의 방법들에 대체 될 수 있는 효과적인 인간 EGF 발현 시스템을 제공하고 있다.

• Journal of Microbiology and Biotechnology
• /
• 제22권3호
• /
• pp.316-325
• /
• 2012

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

• Journal of Microbiology and Biotechnology
• /
• 제29권9호
• /
• pp.1470-1477
• /
• 2019

${\beta}_2$-adrenergic receptor (${\beta}_2-AR$) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ${\beta}$-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and $59.57{\mu}g/l$, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was $3.2{\mu}g/l$ and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ${\beta}_2-AR$ would have great application potential in detection of ${\beta}$-agonist residues.

• Journal of Microbiology and Biotechnology
• /
• 제30권7호
• /
• pp.1082-1091
• /
• 2020

Microbial transglutaminases (MTGs) are widely used in the food industry. In this study, the MTG gene of Streptomyces sp. TYQ1024 was cloned and expressed in a food-grade bacterial strain, Bacillus subtilis SCK6. Extracellular activity of the MTG after codon and signal peptide (SP Ync M) optimization was 20 times that of the pre-optimized enzyme. After purification, the molecular weight of the MTG was 38 kDa and the specific activity was 63.75 U/mg. The optimal temperature and pH for the recombinant MTG activity were 50℃ and 8.0, respectively. MTG activity increased 1.42-fold in the presence of β-ME and 1.6-fold in the presence of DTT. Moreover, 18% sodium chloride still resulted in 83% enzyme activity, which showed good salt tolerance. Cross-linking gelatin with the MTG increased the strength of gelatin 1.67 times and increased the thermal denaturation temperature from 61.8 to 75.8℃. The MTG also significantly increased the strength and thermal stability of gelatin. These characteristics demonstrated the huge commercial potential of MTG, such as for applications in salted protein foods.

• KSBB Journal
• /
• 제32권1호
• /
• pp.16-21
• /
• 2017

The multiple sclerosis caused by multiple inflammatory disease or immune system disorder, is usually treated by interferon ${\beta}$ through adjusting the abnormal immune reactions. For high production of human interferon ${\beta}$ using recombinant E. coli, codon optimized and wild type genes were synthesized. When pET-15b or pET-21a vector was used as an expression vector with each gene, there was no target protein expression. When pQE30 vector was used as an expression vector, human interferon ${\beta}$ was expressed by recombinant E. coli XL1-blue and E. coli JM109. Using the codon optimized gene, the expression of human interferon ${\beta}$ was slightly increased as compared to that from wild type gene. However, most of expressed human interferon ${\beta}$ was insoluble form.

• Journal of Microbiology and Biotechnology
• /
• 제13권3호
• /
• pp.344-347
• /
• 2003

Unnatural amino acid S-(2-nitrobenzyl)cysteine was incorporated into human erythropoietin by using a programmed suppression of nonsense codon in a cell-free protein synthesis system. Several controlling factors affecting the operational efficiency of the suppression were investigated and optimized. The amount of suppressor tRNA and the concentration of $Mg^2+$ were crucial not only for the efficiency but also for the control of the exact suppression. In addition, some general optimization factor are reported in order to improve the efficiency in an unnatural amino acid mutagenesis.

• Journal of Microbiology and Biotechnology
• /
• 제20권3호
• /
• pp.467-473
• /
• 2010

To improve the expression efficiency of recombinant endo-$\beta$-1,4-glucanase in P. pastoris, the endo-$\beta$-1,4-glucanase (egI) gene from Aspergillus niger was synthesized using optimized codons. Fourteen pairs of oligonucleotides with 15 bp overlap were designed and the full-length syn-egI gene was generated by two-step PCR-based DNA synthesis. In the synthesized endo-$\beta$-1,4-glucanase gene syn-egI, 193 nucleotides were changed, and the G+C content was decreased from 54% to 44.2%. The syn-egI gene was inserted into pPIC9K and transformed into P. pastoris GS115 by electroporation. The enzyme activity of recombinant P. pastoris stain 2-7# reached 20.3 U/ml with 1% barley $\beta$-glucan and 3.3 U/ml with 1% carboxymethylcellulose (CMC) as substrates in shake flasks versus 1,270.3 U/ml and 220.7 U/ml for the same substrates in 50-1 fermentors. The molecular mass of the recombinant protein was approximately 40 kDa as determined by SDS-PAGE analysis, the optimal temperature for recombinant enzyme activity was $70^{\circ}C$, and the optimal pH was 5.0 when CMC was used as the substrate.

• Journal of Microbiology and Biotechnology
• /
• 제26권2호
• /
• pp.356-363
• /
• 2016

Human papillomavirus (HPV), a non-enveloped, double-stranded DNA tumor virus, is a primary etiological agent of cervical cancer development. As a potential tool for prophylactic vaccination, the development of virus-like particles (VLPs) containing the HPV16 L1 capsid protein is highly desired. In this study, we developed a high-level expression system of the HPV16 L1 in Escherichia coli for the purpose of VLP development. The native gene of HPV16 L1 has many rare codons that cause the early termination of translation and result in the production of truncated forms. First, we optimized the codon of the HPV16 L1 gene to the preferable codons of E. coli, and we succeeded in producing the full-size HPV16 L1 protein without early termination. Next, to find the best host for the production of HPV16 L1, we examined a total of eight E. coli strains, and E. coli BL21(DE3) with the highest yield among the strains was selected. With the selected host-vector system, we did a fed-batch cultivation in a lab-scale bioreactor. Two different feeding solutions (complex and defined feeding solutions) were examined and, when the complex feeding solution was used, a 6-fold higher production yield (4.6 g/l) was obtained compared with that with the defined feeding solution.