Abstract
Neurotrophin-4 (NT-4) is the most recently discovered member of the neurotrophin family. We have expressed, refolded, and purified recombinant human NT-4 from Escherichia coli and compared it with recombinant human NT-4 secreted into the culture medium of baculovirus-infected insect cells. Both preparations were characterized and determined to be indistinguishable according to several biochemical criteria. Recombinant NT-4 from E. coli was crystallized in a form suitable for x-ray analysis, and characterization of these crystals indicated that NT-4 was present as a dimer within the asymmetric unit. NT-4 was active in promoting the survival of rat TrkB receptor-expressing fibroblasts, but was inactive on embryonic chicken sensory neurons, unlike the other members of the neurotrophin family and in contrast to the reported activities of partially purified NT-4.
Highlights
Characterization and Crystallizationof Recombinant Human Neurotrophind*(Received forpublication, July 21, 1993, and in revised form, September 2, 1993). James P
Neurotrophin-4(NT-4)is the most recently discoveredto the Xenopus NT-4 gene [9, 10]
Biochemical Chamcterization ofRecombinant N72" compare recombinant NT-4 purified from E. coli or from insect cells, we purified protein to greaterthan95% purity fromboth provided by Z
Summary
(Received forpublication, July 21, 1993, and in revised form, September 2, 1993). James P. Neurotrophin-4(NT-4)is the most recently discoveredto the Xenopus NT-4 gene [9, 10]. The human NT-4 gene was member of the neurotrophin familyW. Escherichia coli and comparedit with recombinant hu- cells were reported to stimulate neurite grofrwomth embryonic man NT-4secreted into the cultumreedium of baculovi- chicken dorsal root ganglia (DRG) Both preparations were char-and differentiationof PC12 cells [11]. Recombinant neurotrophins from Escherichia coli has proven to be difficult. LvNxwadii-skiTavrcyes-aaam4lyttfeimhrondoeafaemtnctrhoratataiiElhvctyt.eesoTcurNinrosnaT,lkmeiin-tBm4.wdeNmbrawTcesr-bahcy4cseeaowrrpprnyostaarifsocecstrtstahe-eaclerenlchxiittzzipincaevrekdateeiuseoiisnrnnnsaoiopnatfsdrrgeftooihnfompmiersbheosmorirertnosyiwbnucfgiliratanthyamsetsitbhunstiellar,eytbolhssfnuoueastrnri,n-d-utAiseinhzncitate-rthiptsebieoemoeronsprpatotrsyhsfoimephetleeuoadrxmtstupeiaerroxeensrptsorsNlpeoiNorTswosnT-,t4,-se4pfrpierenpuofcsuormi,irflfdiiiocfr,cieraaeacastdcoinotfmidufnvrsb,oiiaptiomynnunaprd(inp1rfboty5rioEto-e1tc.tei8hhnci)eeno.nmslsieT,eiahucchinarraosledvtctprecehooadaprpmreheaispnircunattldroestee,res--dthe in contrast to the reported activitiesof partially puri- cell culture mediumof recombinant baculovirus-infected insect fied NT-4.
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