Expression, purification, and functional characterization of the serine protease inhibitor neuroserpin expressed in Drosophila S2 cells.
Neuroserpin (NS) is a serine protease inhibitor (or serpin) that is widely expressed in the developing and adult nervous systems. It has been implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration, and axogenesis. To aid in the characterization of this new serpin we have established a high-level expression system in Drosophila S2 cells and developed a purification strategy to obtain neuroserpin for functional studies. Suspension cultures of S2-NS cells secreted recombinant neuroserpin into the medium. High-level expression was maintained when the cells were switched to a nonselection serum-free medium for 3-4 days to facilitate protein purification. Recombinant neuroserpin was purified by sequential chromatography on Macroprep ceramic hydroxyapatite, Type I, POROS HQ20, Resource Q, and Superdex 75 HR 10/30 media. Two secreted forms of neuroserpin were observed with molecular weights of approximately 49 and approximately 50 kDa which may represent alternative glycosylation at three putative N-linked glycosylation sites. Amino acid sequence analysis indicated three NH(2)-terminal sequences. The major sequence was generated by cleavage at the Gly(18)-Ala(19) bond consistent with removal of an 18-amino-acid signal peptide. Two further sequences were identified each with one fewer amino acids at the NH(2)-terminus. All three NH(2)-terminal sequences were also identified by mass spectrometric analysis of neuroserpin following trypsin digestion. Mass spectrometry also confirmed the protein had an intact carboxyl terminus while complex formation assays indicated the inhibitor was functionally active. In summary, Drosophila S2 cells offered a nonlytic stable expression system for the continual production of neuroserpin in high-density suspension cultures.
Hill
R M
RM
Molecular Neuroendocrinology Laboratory, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Brennan
S O
SO
Birch
N P
NP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004331:Drosophila melanogaster / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015843:Serpins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000097522:Neuroserpin
D009479:Neuropeptides; D010960:Plasminogen Activators; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000097522:Neuroserpin