Selective deamidation of recombinant human stem cell factor during in vitro aging: Isolation and characterization of the aspartyl and isoaspartyl homodimers and heterodimers

During in vitro aging, deamidation of recombinant human stem cell factor produced in Escherichia. coli was detected by HPLC analysis and by the release of soluble ammonia. The deamidation rate is very slow in buffers at low pH or at low temperatures; however, the rate is significantly accelerated in alkaline buffers such as sodium bicarbonate in combination with elevated temperatures. HPLC isolation of various deamidated forms followed by peptide mapping and mass spectrometric analyses revealed that the deamidation involves Asn10 in the sequence -T9NNV- near the N-terminus of the protein. Following peptide mapping analysis, significant amounts of aspartyl and isoaspartyl peptides were identified, indicating the conversion of asparagine into both aspartate and isoaspartate residues. As a result of spontaneous association−dissociation of stem cell factor dimer, a total of five deamidated forms, including two homodimers and three heterodimers, were detected and isolated. Cell proliferation assays showed that...