Part:BBa_K3767001

Alkaline Phosphatase optimized for E. Coli w/ 40x catalytic activity

Alkaline phosphatases are a group of isoenzymes that catalyze the hydrolysis of organic phosphate esters present in the extracellular space^[1] . This catalytic activity is commonly used in diagnostic tests as the cleavage of the phosphate ester produces fluorescence visible to the naked eye.

Figure 1: 3D representation of the alkaline phosphatase. A and B domains shown in red and green respectively. Zinc and Magnesium highlighted in grey and blue respectively

This part is being registered as an improvement on a previously registered part BBa_K1216001. Our contribution altered the sequence to improve enzymatic activity and expression rates in E. Coli.

Usage and Biology

Alkaline phosphatase are plasma membrane-bound glycoproteins which are capable of hydrolyzing monophosphate esters resulting in the release of inorganic phosphate^[3]. Dephosphorylation activity is catalyzed by two Zn2+ and one Mg2+ ions within the active site with both ions being required for enzymatic activity. In addition to this, both metal ions aid in structure conformation stability and subunit-subunit interactions.

The varying reaction mechanisms of this enzyme are well-conserved across animal and bacteria variants with either Ser, Thr, or Cys being modified within the active site during transfer (the residue used depends on the activity type)^[4]. Additionally, the final phosphate receptor is conserved being either H2O, a secondary substrate, or another hydroxyl group within the same molecule. An illustration of the transfer of the phosphate group is depicted below.

Figure 2. Reaction mechanism of Alkaline Phosphatase ^[4].

Additional characterisation by Sheffield iGEM 2024

Our team cloned this into a pet21a(+)-BsaI-sfGFP. Protein production strain BL21 Lemo was used and protein was purified via Immobilised Metal Affinity Chromatography (IMAC), and relevant fractions were analysed via SDS-PAGE electrophoresis. PhoA yield was lower than PafA from flavobacterium johnsoniae (BBa_k5172000) but still significant in elutions 4-6 (see figure 3) . Elutions with proteins were pooled and the buffer was exchanged into a glycerol-based buffer.

Figure 3: SDS-PAGE results of PhoA and the PafA post-protein purification. The flow-through (FT), binding buffer (BB), wash buffer (WB), and elutions (E1-10) were all run on the gel to identify which elutions contained protein.

Size exlusion chromatography was done to remove extra banding, signifying other protein impurities.

Figure 4: SDS-PAGE results of PafA WT and PhoA post-SEC.

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

1. Lowe, D., Sanvictores, T., and John, S. (2021) Alkaline Phosphatase, StatPearls Publishing, [online] http://www.ncbi.nlm.nih.gov/pubmed/29083622 (Accessed June 8, 2021)
2. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 8, 2021)
3. Sharma, U., Pal, D., and Prasad, R. (2014) Alkaline phosphatase: An overview. Indian J. Clin. Biochem. 29, 269–278
4. Millán, J. L. (2006) Alkaline phosphatases. Purinergic Signal. 2, 335–341
5. Du, M. H. L., Lamoure, C., Muller, B. H., Bulgakov, O. V., Lajeunesse, E., Ménez, A., and Boulain, J. C. (2002) Artificial evolution of an enzyme active site: Structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953