Part:BBa_K5384007

pPIC9K-His-DDDDK-Vg

Vglycin gene and DDDDK site were linked to plasmid pPIC9K after inserting Vglycin coding polypeptide gene from Pichia pastoris plasmid. We designed the new plasmid in the experiment, because we need to transform Pichia pastoris to express our target protein Vglycin, the plasmid pPIC9K-His-DDDDK-Vg, which we constructed and used, has a replication origin of the Pichia pastoris plasmid, an AOX1 promoter replication promoter, an α-factor secretion signal replication signal, and a plasmid AOX1 terminator replication terminator, it is convenient to insert and express the target gene and screen out the recombinant. The Ori site of the Pichia pastoris plasmid was derived from the Pichia pastoris plasmid.

Figure 1: Visualization of the pPIC9K-his-DDDDK-Vg for overexpression

Usage and Biology

This composite part contains three His-tag tags (BBa_K5384006), the Vglycin gene (BBa_K5384001), the Enterokinase recognition site (BBa_K5384002), the Asp-pro acid-sensitive site (BBa_K5384003), and upstream the AOX1 promoter (BBa_K5384004), and α-secretion signal peptide (BBa_K5384005).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1121
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 142
  Illegal XhoI site found at 1342
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1121
  
• 1000
  COMPATIBLE WITH RFC[1000]

Application

After inserting the Vglycin-encoding polypeptide gene from the Pichia pastoris plasmid, the Vglycin gene and the DDDDK site were ligated to the plasmid pPIC9K. We designed new plasmids in our experiments because we needed to transform Pichia pastoris to express our target protein Vglycin, and we constructed and used the plasmid PIC9K-3His-DDDK-vg with the replication start of the Pichia pastoris plasmid, AOX1 promoter replication promoter, α-factor secretion signal replication signal, and plasmid AOX1 terminator replication terminator, which facilitates insertion and expression of target genes and screening of recombinants. The Ori site of the Pichia pastoris plasmid was derived from the Pichia pastoris plasmid.

References

[1] WANG Wenli, WANG Yunlong, LI Chenyang, et al. Preparation, Identification and Preliminary Application of His-tagged Monoclonal Antibody[J]. J Cell and Molecular Immunol,2008,24(4):399-400. DOI:10.3321/j.issn:1007-8738.2008.04.028.

[2] LI Shuying, ZHAO Zhonglin, NIE Ying, et al. Research Progress on Nattokinase[J]. China Agricultural Science and Technology Review,2013,15(4):139-143.] DOI:10.3969/j.issn.1008-0864.2013.04.21.

[3] Huang Zhili, Luo Lixin, Yang Rude, et al. Nattokinase[J]. Chemistry of Life,2000(2):82-83.] DOI:10.3969/j.issn.1000-1336.2000.02.012.

[4] Zhao Fuyong, Yan Han, Ren Guangxu, et al. Research Progress of Recombinant Nattokinase[J]. China Food and Nutrition,2019,25(7):41-45.] DOI:10.3969/j.issn.1006-9577.2019.07.008. Establishment of nattokinase detection system and a preliminary study on its transmembrane transport pathway[J]. Journal of Chengdu Medical College,2016,11(5):532-536,564. DOI:10.3969/j.issn.1674-2257.2016.05.003.

[5] Tang Xiaoyan. Screening and application evaluation of efficient transcriptional termination sequences in Pichia pastoris[D]. Guangdong:South China University of Technology,2019(in Chinese).