Journal: Applied Microbiology and Biotechnology

Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics

Karin Hjort, Ilaria Presti, Annelie Elväng, Flavia Marinelli and Sara Sjöling

Corresponding author: Sara Sjöling, e-mail: ; Tel. +46-8-6084767; Fax. +46-8-6084510

Electronic Supplementary Material

Heterologous expression of GST-Chi18H8 fusion protein

For heterologous expression in E. coli BL21 (DE3),starter cultures were prepared by growing the pGEX-6P-3::chi18H8 clone over-night at 37 °C in flasks containing Luria-Bertani (LB) broth with 100 µg/mL ampicillin(Sigma-Aldrich, St. Louis, MO, USA). The cultures weresubsequently diluted with 80 mL of LB media to a starting O.D.600 nm of0.1 in 300 mL baffled Erlenmeyer flasks and incubated at 37 °C and 200 rpm. The following liquid media were tested: LB, Terrific Broth (TB), 2× Yeast Tryptone (2×YT), 2×Yeast Tryptone Glucose (2×YTG), Malt Extract (ME) (Sigma-Aldrich). For induction, 0.1 or 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich) was added at two different growth phases (early or late exponential growth phase). After induction, the cells were cultured for different time intervals at different temperatures (16 or 25 °C) at 150 rpm. The cells were harvested by centrifugation at 8000 gfor 15 minutes.The cell pellet was sonicated (6 cycles of 30 seconds each, with a 1 minute interval using a Branson Sonifier 250, Danbury, USA) in Phosphate Buffered Saline (PBS) pH 7.4 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), containing 1 mM phenylmethylsulfonylfluoride (Sigma-Aldrich), 0.7 μg/mL of pepstatin A (Sigma-Aldrich) and 10 μg/mL deoxyribonuclease I (2 mL/g cells) (Sigma-Aldrich). The soluble fraction was separated from the insoluble fraction by centrifugation for 1 hour at 18000 g at 4 °C. Both soluble and insoluble fractions were re-suspended in the same volume of sonication buffer and assayed for chitinase activity by the fluorimetric assaywith 4-methylumbelliferyl-N-N’-chitobiose (MU-NAG2) as substrate.

As showed in Figure S1a, a slight increase in soluble recombinant GST-Chi18H8was achieved when IPTG was added at a concentration of 0.1 mM to an early exponentially growing E. coli pGEX-6P-3::chi18H8.Out of the five media tested, ME improved protein expression inboth soluble and insoluble fractions (Fig. S1b). Decreasing the growth temperature (ME medium) to 16 °C after IPTG induction and prolonging the incubation timeup till 48 hours enhanced the fraction of soluble protein(Fig. S1c).Thus, the procedure finally used to produce soluble recombinant GST-Chi18H8consisted of inducing cells, growing in ME, at early exponential phase with 0.1 mM IPTG and harvesting the cells after 48 hours at 16 °C. Under these conditions, 21 and 51 units of chitobiosidase per liter of culture were detected in the soluble and insoluble fractions, respectively.Protein analysis by SDS-PAGE(Fig. S2) showed a dominant protein fragmentof the expected mass of 73 kDa, corresponding to the fusion protein GST-Chi18H8in the insoluble fraction. This protein fragment was detectable but faint in the SDS PAGE of the soluble fractions. Indeed, the chitobiosidase activity assay indicated a1:2 ratio between soluble and insolubleform of GST-Chi18H8. This discrepancy can be explained by the high sensitivity of the chitobiosidase assay (McCreath and Gooday 1992; Di Maro et al. 2010), and taking into account that most recombinant proteins lose (partially or completely) their biological activity when aggregated in insoluble masses and packed into inclusion bodies in E. coli (Huang et al. 2012). As expected, the 73 kDa protein was not detectable in the control E. coli pGEX-6P-3, which produced a band of 26 kDa corresponding to the free GST protein in the soluble form (Fig. S2).

Fig. S1.

a)

b)

c)

Fig. S1a)Protein expression of Chi18H8. Effect of IPTG induction on GST-18H8 production by E. coli pGEX-6P-3::chi18H8. Two different IPTG concentrations (0.1 or 0.5 mM) were added at two different stages of growth (first or late exponential growth phases). The recombinant strain was grown in LB medium at 37 °C and 200 rpm. After induction, temperature and agitation were decreased to 25 °C and 150 rpm. Cells were harvested three hours (O.D.600 nm = 1-1.5) after the induction. The chitobiosidase activity was measured in the soluble fractions (white bars) and insoluble fractions (black bars) by fluorimetric assay.b) Effect of different growth media on GST-Chi18H8 production by E. coli pGEX-6P-3::chi18H8. The recombinant strain was cultivated as described above and induction was carried out by the addition of 0.1 mM IPTG at the first exponential growth phase. Cells were harvested at an O.D.600 nm of 1. c) Effect of cell harvesting time on GST-Chi18H8 production by E. coli pGEX-6P-3::chi18H8 in ME medium. Recombinant strain was grown at 37 °C and 200 rpm. At an O.D.600 nm of 0.4, induction was carried out by the addition of 0.1 mM IPTG and the temperature was decreased to 16 °C. Cells were collected after 18, 24, 30 and 48 hours from the induction, respectively.

Fig. S2.

Fig.S2. Comparison of proteins in E. coli pGEX-6P-3 and E. coli pGEX-6P-3::chi18H8 insoluble fractions (lane 1 and 2, respectively) and E. coli pGEX-6P-3 and E. coli pGEX-6P-3::chi18H8soluble fractions (lane 3 and 4) by SDS-PAGE analysis. Recombinant strains were grown in ME medium at 37 °C and 200 rpm. At O.D.600 nm of 0.4, induction was carried out by 0.1 mM IPTG and temperaturewas decreased to16 °C. Cells were collected after 48 hours from IPTG induction. Samples loaded in each lane correspond to 1 mL of culture.

References

Di Maro A, Terracciano I, Sticco L, Fiandra L, Ruocco M, Corrado G, Parente A, Rao R (2010) Purification and characterization of a viral chitinase active against plant pathogens and herbivores from transgenic tobacco. J Biotechnol 147:1–6

Huang C, Lin H, Yang X (2012) Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. J Ind Microbiol Biot 39:383–399

McCreath KJ, Gooday GW (1992) A rapid and sensitive microassay for determination of chitinolytic activity. J Microbiol Meth 14:229–237

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