Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Lactic acid bacteria host-vector system for heterologous expression

To verify the possibility of using the S-layer protein of Lactobacillus crispatus M247 as a fusion partner for heterologous gene expression in Gram-positive bacteria, the antiviral polypeptide cyanovirin-N (CV-N) was expressed as fusion with the S-layer in Streptococcus gordonii. For this purpose we included in the genetic fusion the signal sequence and transcription/translational signals of the S-layer gene. Recombinant CV-N/S-layer proteins included different portions of the S-layer fusion partner, and were expressed in the supernatant of recombinant S. gordonii. To construct CV-N/S-layer fusion, the CV-N gene was cloned in frame into the S-layer gene in plasmid pSMB366. Different genetic fusion were constructed, using unique restriction sites within the S-layer for cloning CV-N. The CV-N gene was PCR-amplified from plasmid pelB/pET26b(+) by using two different set of primers and cloned in pSMB366. This plasmid is a derivative of the previously developed vector pSMB47, and contains the S-layer gene with its original promoter and terminator sequence. The integration of this vector into the chromosome of S. gordonii was based on the homologous recombination into the tet(M) gene of the broad range transposon Tn5253 of the recipient strain.

Two recombinant plasmids were obtained, depending on the restriction sites used for cloning, which were named pSMB392 and pSMB393. Both plasmids included the S-layer signal sequence responsible for the export of the protein at the cell surface, and the S-layer transcription/translational signals. These plasmids were used to transform S. gordonii GP201, and transformants were selected. The recombinant S. gordonii strains were subjected to cell fractionation, and culture supernatants were concentrated by filtration. Expression of CV-N/S-layer fusions in recombinant S. gordonii was assayed by Western blot analysis, using both anti-CV-N rabbit serum diluted 1:1000, and anti-S-layer rabbit serum diluted 1:3000. Two recombinant S. gordonii strains were constructed, expressing CV-N as a fusion with different portion of S-layer protein. In Western blot analysis reactive bands were detected in GP1405 and GP1406 culture supernatant, using both anti-CV-N and anti-S-layer rabbit sera. The estimated molecular weights of the bands corresponding to mature proteins produced by GP1405 and GP1406 are 21 and 46 kDa, respectively, according to those deduced from the amino acid sequence (Fig. 1B). Only negligible traces of the recombinant proteins were found in surface associated protein fraction. Both the two fusion proteins, which included different portion of the S-layer, were expressed efficiently in S. gordonii. Therefore, shortening the fusion partner did not affect significantly the expression of recombinant proteins. These results demonstrated that the S-layer of L.crispatus M247 could be used as a fusion partner for heterologous gene expression. This strategy may be widely applicable for expressing heterologous proteins in commensal lactic acid bacteria as a possible approach for local delivery of bioactive molecules.

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Università di Siena,
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