Part:BBa_K4005005

PET28a-rANG

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4402
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2622
  Illegal NgoMIV site found at 2782
  Illegal NgoMIV site found at 4370
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Angiogenin (Angiogenin, ANG) belongs to the secreted ribonuclease superfamily, and was originally discovered to be an angiogenic factor derived from human tumors. Subsequent studies have shown that ANG is also present in normal human tissues and body fluids. ANG increases the transcription of ribosomal RNA (rRNA) in the nucleus, while vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) activate mTOR and S6 kinase pathways to promote protein synthesis. Therefore, ANG can cooperate with VEGF and FGF-2 to promote protein synthesis in endothelial cells. In addition, ANG is necessary for endothelial cell proliferation. In addition to promoting angiogenesis, ANG is also involved in many other physiological and pathological processes, such as neuroprotection, inflammation, and tumorigenesis. Loss of function mutations in the ANG gene have been found in both amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD). According to literature reports, heparan sulphate proteoglycans (HSPGs) are required for ANG to enter cells. HSPGs are linear anionic glycosaminoglycan (GAG) chains covalently linked to the core protein, composed of disaccharide repeating units of uronic acid and glucosamine, and the glucosamine residues 3-OH, 6-OH and -NH And the 2-OH of uronic acid is replaced by a sulfate group. HSPGs exist on the plasma membrane of all animals and are the main component of the extracellular matrix. Factors such as size, location, degree of sulfation, and uronic acid in different cells, tissues, and developmental stages contribute to the structural diversity of heparan sulphate (HS). This structural diversity is the basis for HSPGs to perform multiple functions. In previous research, our laboratory constructed a library of HS mutant mouse lung endothelial cells by knocking out key genes in the HS biosynthesis process. In this study, we prokaryotic expression and purification of recombinant ANG protein, using heparin and heparin derivatives and HS mutant mouse lung endothelial cell library to perform enzyme-linked immunosorbent assay and flow cytometry experiments to explore the interaction between ANG and HS The specificity and clarify its structure-activity relationship. pro-rANG-His-ter

Construct design

Figure 1. The rANG protein expression box..

The profiles of every basic part are as follows:

BBa_K3521004

Usage and Biology

This part is used to effectively express the target protein. It contains T7 promoter and terminator, N-terminal and C-terminal His tags, kanamycin resistance gene, and the replication origin site of E. coli

BBa_K4005000

Usage and Biology

ANG is necessary for the proliferation of endothelial cells, and mutations in the loss of function of the ANG gene are likely to be important causes of cardiovascular diseases such as amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD)

Experimental approach

pro-rANG-His-ter which produces ANG protein is shown in Figure2.

Figure2 : the scanning gel electrophoresis map of the plasmid vector after enzyme digestion.

Figure2 : the scanning gel electrophoresis map of the plasmid vector after enzyme digestion.

As seen in figure 2, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9 is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli transformation.

Figure 3: The gel electrophoresis map of PCR.

Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme digestion was conducted and it was linked with digested pET-28a.

Figure 4. E. coil having the desired pET28a-rANG.

Figure 4. E. coil having the desired pET28a-rANG.

The plates in Fig. 4 (1) showed monoclonals of pET28a-rANG constructs. The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in Fig. 4 (2).

Recombination E. coli

Figure 5: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after).

Figure 5: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was after).

Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band 531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals containing the pET-28a-rANG recombinant plasmid.

Reference

World Health Organization Estimated Deaths 2012.

http://www.who.int/entity/healthinfo/global_burden_disease/GHE_Deaths_2012_country.xls?ua=1

Parkinson disease: MedlinePlus genetics. (n.d.). MedlinePlus-Health Information from the National Library of Medicine.

https://medlineplus.gov/genetics/condition/parkinson-disease/

Zhang J, et al. Nucleic Acids Res. 2002 Mar 1;30(5):1169-75

https://www.ncbi.nlm.nih.gov/Structure/pdb/5EOP