Earticle

Free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels such as biodiesel. Acyl-acyl carrier protein (ACP) thioesterase (acyl-ACP TE) from Streptococcus pyogenes MGAS10270, was codon-optimized and expressed in Escherichia coli K-12 MG1655 and Escherichia coli K-12 W3110. By utilizing codon-optimized S. pyogenes acyl-ACP TE to improve the total FFAs and to tailor the composition of FFAs, high-specificity production of saturated fatty acids (C12, C14) and unsaturated fatty acids (C18:1 C18:2) were achieved in recombinants. E. coli SGJS41 and SGJS46 (codon-optimized acyl-ACP TE of S. pyogenes) demonstrated the highest intracellular total FFAs content; in particular, the content of C12 and C14 FFAs was about 3–5 fold, and the content of C18:1 and C18:2 FFAs content about 8–42 fold higher than that in the control E. coli and E. coli JES1017 (original acyl-ACP TE of S. pyogenes).

본 연구에서는 컴퓨터 기반 예측 프로그램을 활용하여 알라닌 아미노산이 반복적으로 포함된 항결빙 단백질 (antifreeze proteins, AFPs)의 효율적인 생산 시스템을 개발하고자 하였다. 이를 위해 대장균 숙주 세포의 유전자 코돈 과 GC 염기서열 함량 비율을 최적화하는 전략을 적용하였다. 먼저, 다양한 유전자 및 단백질 데이터베이스를 분석하여 여러 종류의 AFP 유전자 중 AFP01과 AFP02를 선정한 후, OptimumGene™ 코돈 최적화 분석을 수행하여 숙주 세포 에 적합하도록 코돈 선호도 및 GC 함량을 조정하였다. 최적화 결과, AFP01 유전자의 코돈 적응 지수(Codon Adaptation Index, CAI)는 0.7에서 0.83으로 개선되었으며, GC 함량은 72.6%에서 65.4%로 조정되었다. 또한, AFP02 유전자의 CAI는 0.64에서 0.84로 증가하였고, GC 함량은 최적화 전 67.5%에서 최적화 후 68.2%로 변경되었 다. 이러한 최적화된 유전자 서열을 기반으로 인공적으로 synAFP01과 synAFP02 유전자를 합성하고, 이를 발현 벡터 시스템에 삽입하여 대장균에서의 단백질 발현을 유도하였다. 그 결과, 두 종류의 synAFPs가 높은 수준으로 생산됨이 분자 수준에서 확인되었다. 본 연구에서 개발된 방법을 통해 합성된 AFPs는 식품 및 의약 산업과 같은 결빙 방지가 필요한 다양한 분야에서 활용될 수 있을 것으로 기대된다.

This study aimed to develop an efficient production system for antifreeze proteins (AFPs) containing repetitive alanine residues by optimizing the codon usage and GC content ratio of Escherichia coli host cell genes using a computer-based predictive program. To achieve this, various gene and protein databases were analyzed to select two AFP genes (AFP01 and AFP02). These genes were then subjected to OptimumGene™ codon optimization analysis to adjust codon preference and GC content according to the host cell requirements. As a result, the codon adaptation index (CAI) of the AFP01 gene was improved from 0.7 to 0.83, and its GC content was modified from 72.6% to 65.4%. Similarly, the CAI of the AFP02 gene was increased from 0.64 to 0.84, and its GC content was adjusted from 67.5% to 68.2%. Based on these optimized gene sequences, synAFP01 and synAFP02 genes were artificially synthesized and inserted into an expression vector system to induce protein expression in E. coli. The results confirmed that both synAFPs were produced at high levels at the molecular level. The AFPs synthesized using this method are expected to have broad applications in industries requiring antifreeze properties, such as the food and pharmaceutical sectors.

Studies have demonstrated that PE_PGRS45 is constitutively expressed under various environmental conditions (such as nutrient depletion, hypoxia, and low pH) of the in vitro growth conditions examined, indicating that PE_PGRS45 protein is critical to the basic functions of Mycobacterium tuberculosis. However, there are few reports about the biochemical function and pathogenic mechanism of PE_PGRS45 protein. The fact that this M. tuberculosis gene is not easily expressed in E. coli may be mainly due to the high content of G+C and the use of unique codons. Fusion tags are indispensable tools used to improve the soluble expression of recombinant proteins and accelerate the characterization of protein structure and function. In the present study, His6, Trx, and His6-MBP were used as fusion tags, but only MBP-PE_PGRS45 was expressed solubly. The purification using His6-MBP tag-specific binding to the Ni column was easy to separate after the tag cleavage. We used the purified PE_PGRS45 to immunize New Zealand rabbits and obtained anti-PE_PGRS45 serum. We found that the titer of polyclonal antibodies against PE_PGR45 was higher than 1:256000. The result shows that purified PE_PGRS45 can induce New Zealand rabbits to produce high-titer antibodies. In conclusion, the recombinant protein PE_PGRS45 was successfully expressed in E. coli and specific antiserum was prepared, which will be followed by further evaluation of these specific antigens to develop highly sensitive and specific diagnostic tests for tuberculosis.

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.

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.