title: The development and application of new crystallization method for tobacco mosaic virus coat protein.
abstract: BACKGROUND
  Although tobacco mosaic virus (TMV) coat protein (CP) has been isolated from virus particles and its crystals have grown in ammonium sulfate buffers for many years, to date, no one has reported on the crystallization of recombinant TMV-CP connecting peptides expressed in E. coli.
  
  
  METHODS
  In the present papers genetically engineered TMV-CP was expressed, into which hexahistidine (His) tags or glutathione-S-transferase (GST) tags were incorporated. Considering that GST-tags are long peptides and His-tags are short peptides, an attempt was made to grow crystals of TMV-CP cleaved GST-tags (WT-TMV-CP32) and TMV-CP incorporated His-tags (WT-His-TMV-CP12) simultaneously in ammonium sulfate buffers and commercial crystallization reagents. It was found that the 20S disk form of WT-TMV-CP32 and WT-His-TMV-CP12 did not form high resolution crystals by using various crystallization buffers and commercial crystallization reagents. Subsequently, a new experimental method was adopted in which a range of truncated TMV-CP was constructed by removing several amino acids from the N- or the C-terminal, and high resolution crystals were grown in ammonium sulfate buffers and commercial crystallization reagents.
  
  
  RESULTS
  The new crystallization method was developed and 3.0 Å resolution macromolecular crystal was thereby obtained by removing four amino acids at the C-terminal of His-TMV-CP and connecting six His-tags at the N-terminal of His-TMV-CP (TR-His-TMV-CP19). The Four-layer aggregate disk structure of TR-His-TMV-CP19 was solved. This phenomenon showed that peptides at the C-terminus hindered the growth of high resolution crystals and the peptides interactions at the N-terminus were attributed to the quality of TMV-CP crystals.
  
  
  CONCLUSIONS
  A 3.0 Å resolution macromolecular crystal of TR-His-TMV-CP19 was obtained and the corresponding structure was solved by removing four amino acids at the C-terminus of TMV-CP and connecting His-tags at the N-terminus of TMV-CP. It indicated that short peptides influenced the resolution of TMV-CP crystals.
authors.affiliation: State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering of Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, Guizhou Province, PR China.
authors.forename: Xiangyang
authors.initials: X
authors.lastname: Li
authors.forename: Baoan
authors.initials: B
authors.lastname: Song
authors.forename: Deyu
authors.initials: D
authors.lastname: Hu
authors.forename: Zhenchao
authors.initials: Z
authors.lastname: Wang
authors.forename: Mengjiao
authors.initials: M
authors.lastname: Zeng
authors.forename: Dandan
authors.initials: D
authors.lastname: Yu
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Linhong
authors.initials: L
authors.lastname: Jin
authors.forename: Song
authors.initials: S
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003460:Crystallization / Q000379:methods*; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology; D016415:Sequence Alignment; D014026:Nicotiana / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins