Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Method for recombinant manufacture of matrix proteins with eucaryotic cells

A new construct of recombinant ameloblastin was produced by serial PCR reactions to generate an end product which contains (a) a C-terminal His tag, (b) the human ameloblastin cDNA encoding for the entire protein, and (c) an insect signal peptide (mellitin, honeybee) at the N-terminal, for efficient secretion of the recombinant ameloblastin protein in the insect cells.

It is expected that this method will give higher yields, allowing production of native ameloblastin, instead of the fusion protein previously used for successful induction of bone formation in vitro and in vivo.

The construct was cloned into the baculovirus vector expression system (BAC-TO-BACTM - a pFASTBAC1TM donor plasmid), for production of the recombinant protein in a eukaryotic system. The expressed protein includes a poly-histidine affinity tag, enabling isolation and purification of the protein using NTA-Ni2+. In the new construct the His tag was placed at the C-terminal of the recombinant protein. Recombinant baculovirus genome was generated by means of transposition of the recombinant baculovirus vector into the bacmid in competent DH10bac (E.Coli) cells. The generation of the recombinant baculovirus and expression of the recombinant protein were carried out according to the BAC-TO-BACTM protocol using Spodoptera frugiperda (Sf9) insect cells.

Plaque purification was performed in order to isolate clones of baculovirus producing rsHAMBN+ for a homogeneous infection and protein expression, as was described for rHAMBN+. The plaque purified viruses were tested for ameloblastin expression by infecting Sf9 monolayers followed by Western blot of the recovered proteins from the medium and from the infected cell lysate.

One purified viral clone was selected for the production of the secreted recombinant ameloblastin protein. Analysis of protein expression was performed in a combined experiment:(a) 40ml suspension cultures were infected at 3 different ratios (1:20, 1:50, 1:200).(b) Cell viability as well as the intra and extra cellular expression of the recombinant ameloblastin protein were analyzed at 24, 36 and 48 hours. The highest expression of the recombinant protein was found to be at 24 hours post infection, secreted to the medium, using infection ratios of 1/20-1/50 (Rosenfeld et al. 2005).

The secreted recombinant ameloblastin protein is presently being purified from the medium using native conditions (no yields available at this time). This secreted recombinant ameloblastin protein should carry the same modifications of the naturally secreted ameloblastin, and contain no signal peptide (after secretion). The His-tag should enable fast one-step purification of the secreted recombinant ameloblastin protein from the medium, using Ni-NTA2+ column.

Similar procedures are also used for manufacture of Amelogenin and Tuftelin.

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Dan DEUTSCH, (Professor)
Tel.: +972-2-6758565
Fax: +972-2-6757307