The development and application of new crystallization method for tobacco mosaic virus coat protein

Virology Journal

Song Yang¹  Linhong Jin¹  Zhuo Chen¹  Dandan Yu¹  Mengjiao Zeng¹  Zhenchao Wang¹  Deyu Hu¹  Baoan Song¹  Xiangyang Li¹ 

[1] State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural bioengineering of Ministry of Education, Guizhou University, Huaxi District, Guiyang 550025, Guizhou Province, P. R China

关键词: Truncated protein;    TMV-CP;    Protein crystals;    Disk form;    Peptides;    His-tags;    GST-tags;   

Others  :  1153086
DOI  :  10.1186/1743-422X-9-279

 received in 2012-02-01, accepted in 2012-10-03,  发布年份 2012

【 摘 要 】

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.

Conclusion

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.

【 授权许可】

   
2012 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

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