title: Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology.
abstract: Towards the targeting of recombinant Thermoanaerobacter thermohydrosulfuricus lipase (TtL) for secretion into the culture medium of Escherichia coli, we have investigated a combination of the archeal lipase gene with a Salinovibrio metalloprotease (SVP2) signal peptide sequence. The SVP2 signal peptide has shown all necessary features of a leader sequence for high level secretion of a recombinant target protein in E. coli. Two sets of primers were designed for amplification of the corresponding gene fragments by PCR. Firstly, the PCR product of the TtL gene with designed restriction sites of SacI and HindIII was cloned into pQE-80L plasmid, named as pQE80L-TtL. Afterwards, the amplified fragment of SVP2 signal peptide with EcoRI and SacI restriction sites was also cloned into pQE80L-TtL and the final construct pQE-STL was obtained. A study on the extracellular expression of recombinant STL revealed that most of the enzyme activity was located in the periplasmic space. Glycine and Triton X-100 were investigated to determine whether the leakage of recombinant STL from the outer membrane was promoted, and it was revealed that glycine has a positive effect. Statistical media optimization design was then applied to optimize the effect of seven factors including glycine, Triton X-100, IPTG, yeast extract concentration, incubation time, induction time, and temperature on the extracellular expression of STL. The optimum conditions for the secretion of the lipase was obtained by incubating recombinant E. coli BL21 cells in the medium supplemented by 1.27% glycine and 24h of incubation in the presence of 0.2mM IPTG concentration.
authors.affiliation: Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz University, Shiraz 71454, Iran.
authors.forename: Roya
authors.initials: R
authors.lastname: Pournejati
authors.affiliation: Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz University, Shiraz 71454, Iran; Institute of Biotechnology, Shiraz University, Shiraz, Iran. Electronic address: karbalaei@shirazu.ac.ir.
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Karbalaei-Heidari
authors.affiliation: Department of Biocatalysis, Institute of Chemistry, Technical University Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000494:pharmacology; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D045726:Metalloproteases / Q000235:genetics*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D017830:Octoxynol / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D046968:Thermoanaerobacter / Q000201:enzymology*
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D017830:Octoxynol; C090528:DNA modification methylase HindIII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D008049:Lipase; D015246:Deoxyribonuclease EcoRI; C024408:endodeoxyribonuclease SacI; D015252:Deoxyribonucleases, Type II Site-Specific; D045726:Metalloproteases; D005998:Glycine
keywords: E. coli expression system; Fusion protein; Heterogeneous protein secretion; SVP2 signal peptide; Thermoanaerobacter thermohydrosulfuricus lipase