Difference between revisions of "Part:BBa K782062"

Latest revision as of 17:47, 26 October 2012

ΔCBD Alginate lyase (mature variant)

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 prefer G-rich. 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. Because the selected alginate lyase consists of two domains, a putative chitin binding like-domain which is in bacterial expression system posttranslationally cleaved off and an alginate lyase domain representing the mature enzyme (Sawabe et al., 2007), we prepared a truncated version of P. elyakovii alginate lyase, abbreviated by chitin binding like-domain.

Figure 1. Schematic representation of the BioBrick part for the mature alginate lyase engineered for secretion from eukaryotic cells. PPT LS denotes preprotrypsin leader sequence, Δ cbd aly is mature 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.

Pseudomonas aeruginosa, a well-known opportunistic pathogen, secretes alginate lyase to degrade the alginate that forms the mucoid matrix composing the bacterial biofilm (Schiller et al., 1993). This alginate lyase might therefore also be used for medical applications to degrade the viscous polysaccharide coat of Pseudomonas in patients with cystic fibrosis.

• The coding sequence for alginate lyase was constructed from 3 gBlocks that were a gift from IDT (Integrated DNA Technology).

Characterization

To estimate the production of constructed alginate lyase, HEK293T cells were transfected with vector containing alginate lyase without chitin binding-like domain 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 nitrocellulose membrane detected by immunoblot using anti-Myc antibodies. Mark corresponding to predicted alginate lyase's size is denoted with arrow.

The enzymatic activity was demonstrated by a decrease of the diameter of alginate beads determined by microscopy. Both P. elyakovii alginate lyases (BBa_K782059 and BBa_K782062) degraded alginate beads, decreasing the beads' diameter by 50 % in comparison to the control.

Figure 3. Enzymatic activity of secreted alginate lyases. Alginate lyases from P. elyakovii and P. aeruginosa (BBa_K782059 and BBa_K782062) were produced and secreted by transfected HEK293T cells. Growth media with secreted alginate lyases was concentrated 50-times with Sartorius Vivaspin 6 concentrators (5 kDa MWCO PES). Alginate beads were incubated in concentrated supernatant. For easier observation under confocal microscope, we added 2000 kDa FITC-dextran (depicted in green) to cell supernatants. Beads' diameters were assessed using Leica LAS Image Analysis software.

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.

Schiller, N., Monday, S., and Boyd, C. (1993) Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J. Bacteriol. 175, 4780–4789.

Sequence and Features