3. Develop Strategy

In this section we will discuss the strategy and major aims of our project.

The first step of our project will be the preparation of the naked mole rat hyaluronan synthase gene (has2), as well as the endogenous B. megaterium gene encoding for UDP-glucose 6-dehydrogenase (UDP-GlcDH), which catalyses the formation of the precursor molecule UDP-glucuronate. For the cloning process of the UDP-glcDH gene, we are going to extract the genomic DNA of B. megaterium cells and subsequently amplify the gene directly from the isolated genomic DNA. In the case of the has2 gene this approach is not possible, because it is necessary to adapt the codon usage of the mammalian has2 gene to ensure an efficient expression in B. megaterium. For that reason we are going to adapt the codon usage of has2 in silico with the online codon adaption tool JCat. Afterwards the gene will be synthesised by Eurofins.

The two genes will be cloned into the shuttle plasmid pSMF2.1 which allows on the one hand the efficient amplification of plasmid DNA in high copy numbers in E. coli cells. On the other hand pSMF2.1 is optimised for expression of recombinant proteins in B. megaterium. For the integration of has2 and UDP-glcDH into the pSMF2.1 plasmid, the genes will be amplified with concomitant addition of appropriate restriction sites. For the cloning procedure of the has2 gene the restriction enzymes MluI and SacI will be used, while the UDP glcDH gene will be integrated by use of SacI and SphI.

In the next step two pSMF2.1 constructs will be designed, the first one containing only the has2 gene with adapted codon usage and the second one containing the has2 gene as well as the endogenous UDP-glcDH gene. Each gene will be integrated with its own ribosomal binding site (RBS), to enable an efficient expression. In figure 1 the planed cloning procedure is schematically shown.

Figure 1: Insertion of hyaloronic acid synthase (HaS) in the vector pSMF2.1

Figure 2: Insertion of UDP-Glucose-Dehydrogenase (UDPGDH) in the vector pSMF2.1-HaS

In the next step of our project we are going to transform B. megaterium cells with the created plasmid constructs to finally express the proteins and produce high molecular mass hyaluronic acid (HMM-HA). As expression host we are going to employ the B. megaterium strain MS941, which was derived from the naturally occurring strain DSM319 by knockout of the neutral protease gene nprM (Wittchen et al., 1995) and therefore well suited for protein expression (Stammen et al., 2007).

Before transformation of MS941 cells the bacterial cell wall has to be degraded. Therefore lysozyme will be used which splits the β 1,4-glycosidic bond between N-acetylmuramic acid and N-acetyl-D-glucosamine, and thereby leads to the formation of competent protoplasts. Afterwards the lysozyme will be removed and the transformation will be carried out. Figure 3 schematically shows the transformation of B. megaterium cells of the strain MS941 with the plasmid pSMF2.1_has2_UDP-glcDH.

Figure 3: Transformation of the vector pSMF2.1-HaS-UDPGDH in B.megaterium

After the successful cloning and transformation procedure the transformed MS941 cells will be incubated in baffled flasks in shakers. For the induction of the protein expression Xylose will be added in 0.5 % concentration (w/v) which will lead to an inhibition of the xylose repressor (XylR) and therefore to an activation of the promotor.

To verify the existence of active Has2 we intend to examine the protein expression, as well as the HMM-HA production. For the protein detection we are going to prepare an SDS-Gel in which the expressed Has2 protein should be detectable after staining with Coomassie Brilliant Blue. Furthermore we are going to examine the protein localisation by expressing an eGFP-tagged variant of Has2, which can be visualised by fluorescence microscopy as well as by Western blot analysis with an anti-eGFP antibody.

For the HMM-HA detection and quantification we are going to measure the viscosity of the media 24 h and 48 h after the induction of protein expression. The viscosity should drastically increase, when HMM-HA will be synthesised, because the molecule is able to bind large amounts of water. For the measurement of viscosity we are going to use the method of rheology.

After successful detection of the HMM-HA we are going to purify it following the protocol of Widner et al. (2005). Additionally we are going to verify the high molecular mass of the produced HMM-HA by pulse field gel electrophoresis as described by Tian et al. (2013). Finally we are going to test the effects of the purified HMM-HA on different human cancer cell lines. In particular we are going to examine the CD44 receptor dependant effects and signal cascades in human skin cancer cell lines by proliferation and cytotoxicity assays.

Lastly we are going to improve the purification protocols, as well as the recombinant HMM-HA production by varying different external parameters like flask form, temperature conditions during incubation, incubation time and shaking speed.

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References

Stammen, S., Müller, B.K., Korneli, C., Biedendieck, R., Gamer, M., Franco-Lara, E., and Jahn, D. (2010). High-Yield Intra- and Extracellular Protein Production Using Bacillus megaterium. Appl. Environ. Microbiol. 76, 4037–4046.

Tian, X., Azpurua, J., Hine, C., Vaidya, A., Myakishev-Rempel, M., Ablaeva, J., Mao, Z., Nevo, E., Gorbunova, V., and Seluanov, A. (2013). High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature 499, 346–349.

Widner, B., Behr, R., Dollen, S.V., Tang, M., Heu, T., Sloma, A., Sternberg, D., DeAngelis, P.L., Weigel, P.H., and Brown, S. (2005). Hyaluronic Acid Production in Bacillus subtilis. Appl. Environ. Microbiol. 71, 3747–3752.

Wittchen, K.D., and Meinhardt, F. (1995). Inactivation of the major extracellular protease from Bacillus megaterium DSM319 by gene replacement. Appl. Microbiol. Biotechnol. 42, 871–877.

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