Part:BBa_K782059
Alginate lyase
Introduction
Alginate lyases are not present in mammalian cells but have been found in bacteria, algae and marine mollusks. It has already been demonstrated that the addition of alginate lyase degraded alginate-poly(L-lysine)-alginate microcapsules (Breguet et al., 2007). Alginate lyases degrade alginate by the reaction of β-elimination and are classified based on their substrates: some prefer M-rich alginate, whereas others like G-rich more. Therefore we selected an enzyme that could degrade both, G- and M-blocks of alginate. We found the alginate lyase from bacteria Pseudoalteromonas elyakovii to be a promising candidate, which can degrade all types of alginate (Sawabe et al., 2007). We replaced the original bacterial signal peptide with the preprotrypsin leader sequence to ensure the efficient secretion from mammalian cells and attached a Myc tag at the C-terminus to facilitate the detection of secreted enzyme.
Figure 1. Schematic representation of the BioBrick part for the alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, aly is alginate lyase coding sequence and Myc indicates Myc peptide tag for detection. E = EcoRI restriction site, X = XbaI restriction site, S = SpeI restriction site, P = PstI restriction site.
Characterisation
To estimate the production and secretion of constructed alginate lyase, HEK293T cells were transfected with vector containing alginate lyase downstream of constitutive promoter. After 48 h of protein production cell lysates were prepared. Western blots of lysates were obtained and produced alginate lyase was detected with anti-Myc antibodies.
Figure 2. Production of alginate lyase in HEK293T cells. Lysates of alginate lyase-producing cells (48 h production) were loaded and ran on SDS-PAGE gel. Proteins were blotted on nitrocelulose membrane detected by immunoblot using anti-Myc antibodies. Mark corresponding to predicted alginate lyase's size is denoted with arrow.
Refrences
Breguet, V., and Stockar, U. (2007) Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate. Biotechnol. Prog. 21, 1223–1230.
Sawabe, T., Takahashi, H., Ezura, Y., and Gacesa, P. (2001) Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase. Carbohydrate Res. 335, 11–21.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 64
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 7
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 85
Illegal NgoMIV site found at 820 - 1000COMPATIBLE WITH RFC[1000]
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