Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli

• Published in: Protein expression and purification Vol. 182; p. 105844
• Main Authors: Dong, Guofu, Zhao, Xi, Guo, Junwang, Ma, Lei, Zhou, Hongmei, Liu, Qi, Zhao, Xuelong, Wang, Changzhen, Wu, Ke
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-06-2021
• Subjects: Autophagy; Autophagy-Related Protein 7 - biosynthesis; Autophagy-Related Protein 7 - chemistry; Autophagy-Related Protein 7 - genetics; Autophagy-Related Protein 7 - isolation & purification; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Functional expression; Gene Expression; HIV-1 - genetics; Human ATG7; Humans; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; tat Gene Products, Human Immunodeficiency Virus - biosynthesis; tat Gene Products, Human Immunodeficiency Virus - chemistry; tat Gene Products, Human Immunodeficiency Virus - genetics; tat Gene Products, Human Immunodeficiency Virus - isolation & purification; Tat peptide; DOPE; Tat peptide; CBB; Functional expression; Escherichia coli; Tat; bl-PI; DDM; Autophagy; hATG7; PE; LB; Human ATG7; POPC; IPTG; SUVs
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2021.105844

The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide (‘GRKKRRQRRR’) can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B–I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B–I and promoted LC3B–I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.
•Functional overexpression of recombinant full-length human ATG7 in Escherichia coli.•The recombinant protein showed high purity and excellent biological function.•Tat peptide (‘GRKKRRQRRR’) could increase the solubilities of human ATG7.