Part:BBa_K4138000

2ABH - Phosphate Binding Protein

Profile

Name: 2ABH

Base pairs: 963 bp

Origin: Escherichia coli

Type: Phosphate-binding protein acts as phosphotransferase

E.C Number: 2.7

Usage and Biology

2ABH is a phosphate binding protein (PBP) found in E. coli. Phosphotransferases catalyze phosphorylation reactions and belong to the enzyme category EC number 2.7. Phosphate-binding proteins are responsible for various biological processes as they play a part in the phosphotransferase system (PTS) and bind phosphate with high affinity^[1], but not sulfate. This represents the specificity of phosphate-binding proteins.The phosphate-specific transport system is based on highly-specific transporters or receptors enabling the extraction of phosphate. At low cellular phosphate concentration, the synthesis of phosphate-binding proteins is triggered. Phosphate-binding proteins are mainly found in the periplasm, where they bind phosphate and undergo a conformational change upon binding. The bound phosphate is then transported to membrane proteins, followed by transport into the cytoplasm. 2ABH is one component of our fusion protein SOPP3-2ABH (BBa_K4138004).

Gibson Assembly, Transformation and Expression

We were able to insert the 2ABH gene into the vector pET-28a(+) vector through a Gibson assembly. The pET-28a(+) vector was transformed into chemically competent E. coli BL21(DE3) gold cells via heat shock. After transformation, the 2ABH-Vector construct was expressed in E. coli BL21(DE3) gold. Medium used for Transformation: LB-medium with the addition of kanamycin Expression: TB-medium with the addition of kanamycin, IPTG-induced

Since 2ABH is an E. coli native protein, expression was performed at 37°C.

The additional primers used are listed below:

pET-28a(+) forward primer: BBa_K4138010

pET-28a(+) backward primer: BBa_K4138011

2ABH forward primer: BBa_K4138008

2ABH backward primer: BBa_K4138009

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 645
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Z. Wang, A. Choudhary, P.S. Ledvina, F.A. Quiocho, Fine tuning the specificity of the periplasmic phosphate transport receptor. Site-directed mutagenesis, ligand binding, and crystallographic studies., Journal of Biological Chemistry, Volume 269, Issue 40, 1994, Pages 25091-25094, https://doi.org/10.1016/S0021-9258(17)31503-X.