Characterization of Recombinant Human Brain-Derived Neurotrophic Factor Variants Khurram M. Sunasara,* Steven M. Cramer,* Charles R. Hauer,† Randall G. Rupp,‡ and Vernon A. Shoup‡ ,1 *Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180; †Wadsworth Centre, NY State Department of Health, Albany, New York 12201; and ‡Regeneron Pharmaceuticals Inc., 81 Columbia Turnpike, Rensselaer, New York 12144 Received July 8, 1999, and in revised form September 15, 1999 The purpose of this work was the chemical charac- terization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the ques- tion of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF vari- ants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at po- sitions 31 and 61, respectively. Peak_4, which is chro- matographically a single peak, contained three vari- ants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methio- nine at position 92. Peak_5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB re- ceptor-ligand binding domain of the variants. © 1999 Academic Press Key Words: BDNF activity; norleucine incorporation; methionine sulfoxide; Glu-C digestion; CNBr; homo- serine lactone. Neurotrophins are proteins responsible for the growth, survival, and maintenance of neuronal cells (1–3). The first neurotrophin, nerve growth factor (NGF), 2 was discovered about 40 years ago (4) and its structure–function relationship has since been investi- gated (5–7). The second neurotrophin, brain-derived neurotrophin factor (BDNF), was purified from pig brain by Barde and coworkers a decade ago and sub- sequently shown to be similar to NGF (8). BDNF and NGF have been shown to prevent naturally occurring neuronal death during development (1–3) and are among the two most widely studied neurotrophins. BDNF has been shown to promote the survival and differentiation of motor neurons in vitro for rats (9) and in vivo for rats (10 –12) and chickens (13). BDNF is a basic dimeric protein, the subunit of which is 13.5 kDa (119 amino acids) with a pI of 10.3 (14). Crystal struc- ture studies have shown BDNF to be greatly stabilized by three intrachain disulfides that form an “knot” as shown in Fig. 1 (15, 16). The sequence of BDNF is strongly conserved as identical sequences have been reported for porcine (17), rat (18, 19), mouse (20), and human BDNF (14, 21). BDNF, after being expressed in Escherichia coli. in large scale, shows the presence of variants in the pu- rified pharmaceutical. The high degree of conservation of this growth factor among various species prompted us to characterize the chemical modifications in each of these variants. Furthermore, by comparing the in vitro biological activities of each of these variants with ref- erence BDNF, we can get an insight into whether any of the above chemical modifications are significantly affecting the properties of these variants. The separa- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (518) 488-4030. E-mail: vernon.shoup@regpha.com. 2 Abbreviations used: NGF, nerve growth factor; BDNF, brain- derived neurotrophin factor; TFA, trifluoroacetic acid; PTH, phenyl- thiohydantoin; ACN, acetonitrile; HT-RPLC, high temperature re- versed phase liquid chromatography. 248 0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 372, No. 2, December 15, pp. 248 –260, 1999 Article ID abbi.1999.1501, available online at http://www.idealibrary.com on