Table 1.

Summary of Atg8–PE protein-based in vitro biochemical approaches.

Approach	Mechanism	Required components	Product	Pros	Cons	Applications for autophagy studies	Ref
Enzyme-mediated reconstitution system	Mimicking Atg8 lipidation using purified components and liposomes	Recombinant Atg8-I, Atg7 and Atg3, PE-containing liposomes or giant unilamellar vesicles (GUVs), ATP	Native Atg8–PE	1. Standard and effective approach for in vitro Atg8 lipidation 2. Native Atg8–PE products	Time-consuming and laborious	1. Analyzing Atg8-mediated membrane dynamics 2. Determining influential factors involved in Atg8–PE conjugation 3. Investigating Atg8–PE deconjugation mediated by Atg4	[30,69]
Chemically defined reconstitution system	Maleimide-thiol coupling: Michael addition of the thiol group of cysteine and maleimide moiety	Recombinant LC3BG120C mutant, PE-maleimide-containing liposomes	LC3B–PE maleimide conjugate	1. Simple and time-effective approach for in vitro LC3 lipidation 2. Bypassing the requirement for the conjugation machinery	The product is non-native LC3–PE conjugate	Analyzing LC3-mediated membrane dynamics	[31]
	PolyHis-NTA strategy: high-affinity binding of polyHis-tagged proteins to Ni-NTA	Recombinant LC3B-His12, DGS-NTA-containing liposomes	LC3B-DGS noncovalent complex		1. The commercial DGS-NTA lipids are not native PE structures 2. The product is LC3-DGS noncovalent complex	Analyzing LC3-mediated membrane dynamics	[70]
Cell-free lipidation system	Mimicking LC3 lipidation using purified substrates, fractionated cytosols, membranes and nucleotides.	Recombinant T7-LC3B-I, the cytosols from WT MEFs, the membranes from atg5 knockout MEFs, ATP	Native LC3B–PE	1. Enabling the LC3–PE conjugation in the physiological lipid composition 2. Native LC3–PE products	1. Time-consuming and laborious process, technical challenge 2. Difficult to separate target compartments	1. Defining the origin source of autophagic membranes 2. Dissecting the molecular mechanisms of early autophagic membrane generation	[32,33]
Protein chemical synthesis strategy	PolyArg-assisted solubilization strategy: EPL of LC3[1-114] protein thioester and PE-modified peptide with polyArg tag	LC3B[1-114] MESNa thioester, PE-modified peptide with polyArg tag, MPAA (catalyst)	Native LC3B–PE	Enabling functional LC3–PE in preparative amounts	1. Time-consuming and laborious, technical challenge 2. Need to remove polyArg tag and to refold proteins after ligation	Analyzing LC3-mediated membrane tethering and fusion	[29]
	MBP-assisted solubilization strategy: EPL of MBP-LC3[1-114] protein thioester and PE-modified peptide	MBP-LC3B[1-114] MESNa thioester, PE-modified peptide, β-octylglucoside (detergent), MPAA (catalyst)	Native LC3B–PE	1. Enabling functional LC3–PE with various mutants and modifications in preparative amounts 2. The ligation works under folding conditions	Time-consuming and laborious process, technical challenge	1. Analyzing LC3-mediated membrane tethering and fusion 2. Elucidating the biochemical mechanisms of Legionella RavZ-mediated LC3–PE deconjugation	[28,71]