Difference between revisions of "Part:BBa K4497025"

Latest revision as of 15:59, 13 October 2022

Kappa CL Tetramer

This protein was used to test the previously [1] shown pH-sensitive auto-cleavage sequence (EAAAK)5. We did not observe the cleavage function in our test protein.

Design & Cloning

Design

The part is made of the following components:

• IL27alpha signaling sequence (Part:Bba_K4497009): Signal for protein secretion
• Kappa CL (Part:Bba_K4497001): used simply as the protein to be fused by the linker
• pH linker (EAAAK)5 ((Part:Bba_K4497000)
• 6x His-tag (Part:Bba_K4497026): for purification of the protein using IMAC

Figure 1. Schematic Structure of the pH-sensitive Linker Protein. The IL27alpha signaling sequence for secretion is followed by two Kappa CL domains, the pH-sensitive linker (EAAAK)5, two Kappa CL domains and a 6x His-Tag for purification.

Cloning

The part was ordered inside pcDNA3.4 from GeneArt (Thermofischer).

Purification

The protein was expressed in ExpiCHO using the standard protocol (8days) in 200mL. The supernatant was collected and purified using IMAC followed by size-exclusion chromatography. After purification, relevant collected fractions were analyzed by SDS PAGE. The chosen fractions were pooled, concentrated and frozen using liquid nitrogen. Protein aliquots were kept in a -80 freezer.

Figure 2. 12 % SDS-Gel of pH linker purification. The pH linker was purified with Immobilized Metal Affinity Chromatography (IMAC) and size-exclusion chromatography. Fractions 31 to 38 were determined with IMAC chromatograms at n = 280 nm, reduced samples were loaded (red. 31 to 38). Marker (M) ‘PageRuler Plus Prestained’ was used.

Results

The previously shown pH induced cleavage function[1] of the plasmid was investigated between pH7.5 and pH6.0. The protein showed no pH sensitive auto-cleavage properties. We tested its stability at pH values from 7.5 to 6 at 37°C overnight (Fig. 3). No cleavage products from our 58 kDa sized protein are visible. Given the position of the linker, we would have expected protein fragments from 23 to 25 kDa. 

Figure 3. SDS Gel of pH linker cleavage experiment.pH linker was loaded right after buffer change (0) and after incubation overnight at 37 °C (1) at buffer pH values 7.5, 7.0, 6.8, 6.5, 6.2 and 6.0. Marker (M) ‘PageRuler Plus Prestained’ was loaded.

There could be different reasons for the (EAAAK)5 linker not showing any cleavage activity. For one the cleavage activity might not only be related to the linker itself but also parts of the connected proteins used by Wang et al. Furthermore, this auto-catalytic cleavage might be dependent on other outside factors not shown in the paper. Overall, the available characterization of the cleavage function is quite limited and has not been properly reproduced in other literature. If there are certain other conditions needed for the auto-catalytic cleavage to occur, the usability in cell culture/ in vivo conditions is probably low.

Sequence and Features

References

[1] Wu, Y. J., Fan, C. Y., & Li, Y. K. (2009). Protein purification involving a unique auto-cleavage feature of a repeated EAAAK peptide. Journal of Chromatography B, 877(31), 4015–4021. https://doi.org/10.1016/J.JCHROMB.2009.10.009