title: A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies.
abstract: BACKGROUND
  Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology.
  
  
  RESULTS
  In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo.
  
  
  CONCLUSIONS
  To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.
authors.forename: Govindan
authors.initials: G
authors.lastname: Raghunathan
authors.forename: Ganapathiraman
authors.initials: G
authors.lastname: Munussami
authors.forename: Hyojin
authors.initials: H
authors.lastname: Moon
authors.forename: Hyun-jong
authors.initials: HJ
authors.lastname: Paik
authors.forename: Seong Soo A
authors.initials: SS
authors.lastname: An
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Sebyung
authors.initials: S
authors.lastname: Kang
authors.affiliation: Department of Chemical Engineering, Pusan National University, Busan, South Korea. sungulee@pusan.ac.kr.
authors.forename: Sun-Gu
authors.initials: SG
authors.lastname: Lee
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D002479:Inclusion Bodies / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D053758:Nanoparticles / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C064045:delta protein; D049452:Green Fluorescent Proteins