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  Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

Klatt, S., & Konthur, Z. (2012). Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae. Microbial Cell Factories, 11: 11:97. doi:10.1186/1475-2859-11-97.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-000E-F05C-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-000E-F05D-E
Genre: Journal Article

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© 2012 Klatt and Konthur; licensee BioMed Central Ltd
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 Creators:
Klatt, Stephan1, 2, Author              
Konthur, Zoltán1, Author              
Affiliations:
1In vitro Ligand Screening (Zoltán Konthur), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, Berlin, Germany, ou_1479653              
2Freie Universität Berlin, Faculty of Biology, Chemistry and Pharmacy, Berlin, Germany, ou_persistent22              

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Free keywords: Amino Acid Sequence Gene Expression Humans Leishmania/*genetics/*metabolism Molecular Sequence Data Protein Engineering Protein Processing, Post-Translational *Protein Sorting Signals Protein Transport Recombinant Fusion Proteins/chemistry/genetics/metabolism *Secretory Pathway Single-Chain Antibodies/*chemistry/genetics/*metabolism
 Abstract: BACKGROUND: Secretory signal peptides (SPs) are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1) of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv's). The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. RESULTS: We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv's, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv's contained one additional amino-acid (AA). CONCLUSIONS: The obtained results demonstrate the importance of SP-sequence optimization for efficient expression-secretion of scFv's. We could successfully demonstrate that minor modifications in the AA-sequence in the c-region of the natural SP from SAP1, based on in-silico predictions following the (-3, -1) rule, resulted in different expression-secretion rates of the protein of interest. The yield of scFv production could be improved close to one order of magnitude. Therefore, SP-sequence optimization is a viable option to increase the overall yield of recombinant protein production.

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Language(s): eng - English
 Dates: 2012-07-25
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1186/1475-2859-11-97
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Title: Microbial Cell Factories
Source Genre: Journal
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Publ. Info: BioMed Central
Pages: - Volume / Issue: 11 Sequence Number: 11:97 Start / End Page: - Identifier: ISSN: 1475-2859
CoNE: https://pure.mpg.de/cone/journals/resource/111041294029026