Polysialic acid biosynthesis and production in Escherichia coli: current state and perspectives
Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.