Difference between revisions of "Part:BBa K3187028"

Profile

Name	Sortase A7M
Base pairs	470
Molecular weight	17.85 kDa
Origin	Staphylococcus aureus, synthetic
Parts	Basic part
Properties	calcium-independent, transpeptidase, linking sorting motif LPXTG to poly-glycine Tag

Usage and Biology

Sortase A7M (SrtA7M) is a calcium-independent transpeptidase derived from the enzyme Sortase A from Staphylococcus aureus. ^[1] Similar to Sortase A, SrtA7M connects two peptides covalently, one with a poly-glycine (poly-G) at the N-terminus and one with LPXTG motif at the C-terminus. It acts by cleaving between Gly and Thr of the LPXTG motif and catalyzing the formation of an amide bond to the poly-G sequence. TEST ^[2]

Methods

The basic part Sortase A7M is produced and purified as the composite part BBa_K3187005 (Sortase A7M). For the expression and purification of SrtA7M, a part containing T7 promoter, a lac operator and a ribosomal binding site BBa_K3187029 N-terminal and a 6xHis-Tag with a TGA stop codon (BBa_K3187024) C-terminal of the coding sequence were added. The production was performed in the E. Coli strain BL21 (DE3). The purification was done with the GE Healthcare ÄTKA Pure machine which is a machine for FPLC.

Results

Yield

For the characterization of Sortase A7M an assay was designed to show the coupling efficiency between the TAMRA-LEPTG and the tetrapeptide GGG-Beta-Alanin (GGGßA) catalyzed by the Sortase. The ß-Alanin functions as a spacer. The Sortase reaction was performed for 1h at 30°C and was stopped by enzyme separation through centrifugal filtration. For analysis mass spectrometry (ESI-MS) was used. The mass spectrometry enables differentiation between products and educts. It allowed us to make an estimate of the product yield.

The calculated theoretical molecular masses are 1054 g/mol for TAMRA and 1240 g/mol for TAMRA-LPETGGGßA. Therefore peaks are expected at mass/n, with n ∈ N. By comparison of the number of corresponding peaks estimation of the product yield is possible as both molecules possess the same amount of ionizable groups and thus the difference in the ionizability of both molecules is negligible.

In the Fig. 5 the 621.56 peak can be assigned to the TAMRA-LEPTGGGßA and the 528.85 to the TAMRA-LPETG. The count ratios on the Fig. 1 of the two molecules mentioned show an excess of the product. In this case the Sortase A7M has a coupling efficiency in the range of 80%.

For further characterization an ELISA assay is accomplished to test the efficiency of Sortase A7M especially the impact of the Ca²⁺-dependency. Therefore a piece of paper functionalized with GGGßA is connected to a protein domain, which binds antibodys, with the LPTEG-tag. Negative controls are run to check if the sortase reaction works.

In well 1 there is additional Ca²⁺. In contrast to well 1 in well 2 no Ca²⁺ is added. In well 3 there is no Sortase. The highest absorption can be measured in well 2. This shows that the Sortase is functional with different substrates and that the Sortase A7M is more effective in absence of calcium. The absorption is also relatively high for the negative control, which can be explained by poor washing before the substrate for HPR is added. This assay shows the functionality of Sortase A7M even in context of surfaces.

Primary Amines

This assay was performed to confirm that our variant of the sortases, the Sortase A7M is able to fuse a LPETG-tag with any primary amine independent from the amino acid that is following. It should still prefer the polyG-tag but it could increase the modularity, if the Sortase A7M is also able to connect any primary amine to the LPETG-tag. In this case only the Sortase A7M has been tested. The substrates were 5- Carboxytetramethylrhodamine (TAMRA) with a KLPETG bound to TAMRA via the lysine side chain and 3-azidopropanamine as the example for a primary amine. The reaction was performed for two hours at 37 °C. It was then analyzed by ESI-MS.

Fig.6 shows the educt-peak in the mass spectrum. TAMRA with the LPETG-tag weighed 1054 g/mol. Shown above in green is the single loaded molecule at 1054,27 g/mol and the double loaded molecule at 528,75 g/mol.

Fig.7 shows the product-peak in the mass spectrum. The primary amine that was taken as an example has a molecular weight of 100 g/mol. After the reaction the glycine of the LPETG-tag has been removed and therefore the product only consists of TAMRA-KLPET-3-azidopropanamine. When adding the two molecular weights and subtracting the weight of the glycine it adds up to a total weight of 1078 g/mol which can be seen in the single loaded 1079,37 g/mol peak (Fig. x), since the ESI-MS we used has a small error margin. The peak in black again is the double loaded peak at 541,55 g/mol. This clearly shows that the sortase reaction took place. Furthermore, we can conclude that the Sortase A7M accepts any primary amine as a substrate. However, mass spectrum does not show the ratio of educt and product, which is why we cannot estimate whether the turnover is as high as when using a polyG-tag as substrate.

References

1. ↑ Hee-Jin Jeong, Gita C. Abhiraman, Craig M. Story, Jessica R. Ingram, Stephanie K. Dougan, Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling, PLOS ONE, 2017 [1]
2. ↑ Hongyuan Mao, Scott A. Hart, Amy Schink, and Brian A. Pollok, Sortase-Mediated Protein Ligation: A New Method for Protein Engineering, JACS, 2004 [2]