Yoo Eunhui, Choi Hee Jung, Kim Jin-Kyoo, Kim Young Min, Park Jin Se, Han Jae Yong
Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
Department of International Agricultural Technology & Institute of Green BioScience and Technology, Seoul National University, Pyeongchang, 25354, Gangwon-do, Republic of Korea.
J Biol Eng. 2024 May 7;18(1):32. doi: 10.1186/s13036-024-00427-2.
Adiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems, have faced challenges in generating sufficiently active middle molecular weight (MMW) and high molecular weight (HMW) forms of recombinant human ADPN (hADPN). In our previous study, we proposed genome-edited chickens as an efficient platform for producing multimeric hADPN. However, the consistency of multimeric hADPN expression in this system across generations had not been further investigated.
In this study, subsequent generations of ovalbumin (OVA) ADPN knock-in chickens showed stable multimeric hADPN production, yielding ~ 26% HMW ADPN (0.59 mg/mL) per hen. Comparative analysis revealed that egg white (EW)-derived hADPN predominantly consisted of hexameric and HMW forms, similar to serum-derived hADPN. In contrast, hADPN obtained from human embryonic kidney (HEK) 293 and High-Five (Hi-5) cells also exhibited the presence of trimers, indicating variability across different production systems. Furthermore, transcriptional expression analysis of ADPN multimerization-associated endoplasmic reticulum chaperone genes (Ero1-Lα, DsbA-L, ERP44, and PDI) indicated upregulation in the oviduct magnum of ADPN KI hens, suggesting the chicken oviduct magnum as the optimal site for HMW ADPN production. Lastly, the functional analysis demonstrated that EW-derived hADPN significantly reduced lipid droplets and downregulated lipid accumulation-related genes (LOX-1, AT1R, FAS, and FABP4) in human umbilical vein endothelial cells (HUVECs).
In summary, stable and functional multimeric hADPN can be produced in genome-edited chickens even after generations. This highlights the potential of using chicken bioreactor for producing various high-value proteins.
脂联素（ADPN）在内分泌和心血管功能中起关键作用，但传统的生产方法，如大肠杆菌和哺乳动物系统，在产生足够活性的重组人ADPN（hADPN）中分子量（MMW）和高分子量（HMW）形式方面面临挑战。在我们之前的研究中，我们提出基因编辑鸡作为生产多聚体hADPN的有效平台。然而，该系统中多聚体hADPN跨代表达的一致性尚未进一步研究。
在本研究中，后续世代的卵清蛋白（OVA）ADPN基因敲入鸡显示出稳定的多聚体hADPN产生，每只母鸡产生约26%的HMW ADPN（0.59 mg/mL）。比较分析表明，蛋清（EW）来源的hADPN主要由六聚体和HMW形式组成，类似于血清来源的hADPN。相比之下，从人胚肾（HEK）293和High-Five（Hi-5）细胞获得的hADPN也显示存在三聚体，表明不同生产系统之间存在差异。此外，ADPN多聚化相关内质网伴侣基因（Ero1-Lα、DsbA-L、ERP44和PDI）的转录表达分析表明，ADPN基因敲入母鸡的输卵管峡部上调，表明鸡输卵管峡部是HMW ADPN产生的最佳部位。最后，功能分析表明，EW来源 的hADPN显著减少人脐静脉内皮细胞（HUVECs）中的脂滴，并下调脂质积累相关基因（LOX-1、AT1R、FAS和FABP4）。
总之，即使经过多代，基因编辑鸡也能产生稳定且具有功能的多聚体hADPN。这突出了使用鸡生物反应器生产各种高价值蛋白质的潜力。
