Immunotoxins and Neurotrophins: Novel Strategies for the Efficient Expression of Recombinant Proteins

  • The development of recombinant DNA techniques opened a new era for protein production both in scientific research and industrial application. However, the purification of recombinant proteins is very often quite difficult and inefficient. Therefore, we tried to employ novel techniques for the expression and purification of three pharmacologically interesting proteins: the plant toxin gelonin; a fusion protein of gelonin and the extracellular domain of the subunit of the acetylcholine receptor (gelonin-AchR) and human neurotrophin 3 (hNT3). Recombinant gelonin, acetylcholine receptor a subunit and their fusion product, gelonin-AchR were constructed and expressed. The gelonin gene, a 753 bp polynucleotide was chemically synthesized by Ya-Wei Shi et al. and was kindly provided to us. The gene was first inserted into the vector pUC118 yielding pUC-gel. It was subsequently transferred into pET28a and pET-gel was expressed in E. coli. The product, gelonin was soluble and was purified in two steps showing a homogeneous band corresponding to 28 kD on SDS-PAGE. The expression of the extracellular domain of the -subunit of AchR always led to insoluble aggregates and even upon coexpression with the chaperonin GroESL, very small and hardly reproducible amounts of soluble material were formed, only. Therefore, recombinant AchR- gelonin was cloned and expressed in the same host. The corresponding fusion protein, gelonin-AchR, again formed aggregates and it had to be solubilized in 6 M Gu-HCl for further purification and refolding. The final product, however, was recognized by several monoclonal antibodies directed against the extracellular domain of the -subunit of AchR as well as a polyclonal serum against gelonin. Expression and purification of recombinant hNT3 was achieved by the use of a protein self-splicing system. Based on the reported hNT3 DNA sequence, a 380 bp fragment corresponding to a 14 kD protein was amplified from genomal DNA of human whole blood by PCR. The DNA fragment was cloned into the pTXB1 vector, which contains a DNA fragment of intein and chintin binding domain (CBD). A further construct, pJLA-hNT3, is temperature-inducible. Both constructs expressed the target protein, hNT3-intein-CBD in E. coli by the induction with IPTG or temperature, however, as aggregates. After denaturation and renaturation, the soluble fusion protein was slowly loaded on an affinity column of chitin beads. A 14 kD hNT3 could be isolated after cleavage with DTT either at 4 °C or 25 °C for 48 h. Based on nerve fiber out-growth of the dorsal root ganglia of chicken embryos, both, hNT-3-intein-CBD and hNT3 itself exhibit almost the same biological activity.
  • Immunotoxins and Neurotrophins: Novel Strategies for the Efficient Expression of Recombinant Proteins

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Metadaten
Verfasserangaben:Zhuoyu Li
URN (Permalink):urn:nbn:de:bsz:386-kluedo-12856
Betreuer:Wolfgang E. Trommer
Dokumentart:Dissertation
Sprache der Veröffentlichung:Englisch
Jahr der Fertigstellung:2002
Jahr der Veröffentlichung:2002
Veröffentlichende Institution:Technische Universität Kaiserslautern
Titel verleihende Institution:Technische Universität Kaiserslautern
Datum der Annahme der Abschlussarbeit:08.03.2002
Datum der Publikation (Server):09.04.2002
Freies Schlagwort / Tag:acetylcholine receptor ; gelonin ; immunotoxins ; myasthenia gravis; neurotrophin 3 ; ribosome-inactivating proteins
Fachbereiche / Organisatorische Einheiten:Fachbereich Chemie
DDC-Sachgruppen:5 Naturwissenschaften und Mathematik / 54 Chemie / 540 Chemie und zugeordnete Wissenschaften
Lizenz (Deutsch):Standard gemäß KLUEDO-Leitlinien vor dem 27.05.2011

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