This comb-like polyampholitic polyelectrolyte was synthesized via controlled radical polymerization initiated by the oligoperoxide metal complex (OMC) in a polar organic media. OMC was coordinating Cu²⁺ complex of the copolymer composed of vinyl acetate, 5-tertbutylperoxy-5-methyl-1-hexene-3-yne, and maleic anhydride. Both the initial oligoperoxide and OMC derivate have been synthesized, as previously described (37-39).

Synthesis of the comb-like polyelectrolyte was carried out as follows: a monomer mixture of DMAEM (28.18 g (161.11 mol/l)) and VEP (3.19 g (15.31 mol/l)) was injected into round bottom glass reactor equipped with impeller mixer and backflow condenser. A solution of OMC (1.66 g (0.78 mol/l) in ethanol or dimethyl formamide (67 g (822.8 mol/l)) was then added with stirring. The incubation temperature was increased to 333 K, and the reactor was actively maintained at this temperature for 8 hrs under argon flow. The monomer conversion was controlled using a gravimetric technique; after achieving the desired degree of conversion (i.e., 60%), the solvent was evaporated under vacuum. Synthesized comb-like polyelectrolyte was purified by multiple precipitations from acetone solution into hexane, and dried under vacuum.

Synthesized polymer was dissolved in sterile distilled H₂O, and the pH was adjusted to pH 7.4 (if not mentioned otherwise). A 1% water polymer solution was aliquoted into 1.5 mL plastic tubes (leaving as less air in the tube as possible), and stored at 4°C.

Yeast strains, growth conditions, and media

Hansenula polymorpha NCYC 495 leu1–1, Pichia pastoris GS115 his4 and Saccharomyces cerevisiae BY4742 MATα his3Δ leu2Δ lysΔ ura3Δ strains were grown in YPD medium (1% yeast extract (Serva, Germany), 2% bacto-peptone (Serva, Germany), 2% glucose (Serva, Germany)) at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae), correspondingly.

Minimal modified Burkholder medium was used for selection of P. pastoris and S. cerevisiae transformants (40). For the selection of H. polymorpha transformants via geneticin (G418) resistance, 50 mg/L of G418 (Invitrogen, Sweden) was added to YPD plates. For the auxotrophic strains, amino acids L-leucine, L-uracil, L-histidine, L-lysine (Sigma-Aldrich, USA) were added to a final concentration of 40 mg/L.

DNA manipulation and transformation of Escherichia coli were carried out according to standard procedures (41). E. coli strains were grown in Luria-Bertani medium (LB) at 37°C supplemented with ampicillin (100 µg/mL, Sigma-Aldrich, USA), if necessary.

EIectroporation assay

Electroporation was carried out by using a Bio-Rad Gene Pulser II, USA equipped with a Bio-Rad Pulse Controller II, USA. For yeast electroporation, a modified protocol from Becker and Guarente was used (4).

Transformants were counted 3–5 days after electroporation and statistical analysis (Student's t-test) was performed.

Lithium acetate transformation

A modified protocol for delivering DNA into yeast cells following treatment with lithium acetate (LiAc) was used (30). After 3–5 days of growth, yeast transformants were counted and statistical analysis (Student's t-test) was performed.

Yeast transformation using the developed polymer

H. polymorpha, P. pastoris and S. cerevisiae yeasts were grown overnight in non-selective YPD medium at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae). 150 µL of the overnight culture was transferred into 30 mL of YPD medium and grown until an OD₆₀₀ ≤0.4–0.5. The cells were collected by centrifugation for 10 min at 3000 xg and then re-suspended in 100 µL of YPD. One µL of 1% water solution (pH 7.4) of oligoelectrolyte-based carrier BG-2 and 1 µg of plasmid DNA were added to the cell suspension, mixed gently and kept on ice for 45 min. Subsequently, cells were heat-shocked for 60–90 s at 42°C (H. polymorpha, S. cerevisiae) or 55°C (P. pastoris), chilled on ice for 2 min, and mixed with 1 mL of YPD medium. After incubation at 37°C (H. polymorpha) or 30°C (P. pastoris, S. cerevisiae) for 1 h, 100 µL of cells were plated on a selective medium and incubated at 37°C or 30°C, respectively. Yeast transformants were counted after 3–5 days of growth, and statistical analysis (Student's t-test) was applied.

Stable transformation of Hansenula polymorpha

Yeast transformants were obtained using the protocol employed for transient transformation. After recovering and counting of the transformants, cells were re-plated several times on either selective medium or non-selective medium and grown as described for transient transformation. Stably transformed clones were counted.