scholarly journals Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Innanurdiani Koko ◽  
Adelene Ai-Lian Song ◽  
Mas Jaffri Masarudin ◽  
Raha Abdul Rahim
Keyword(s):  
Signal Peptide ◽  
Heterologous Protein ◽  
Expression System ◽  
Surface Display ◽  
Host Genome ◽  
Integration System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Integrative Vectors

Abstract Background Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901–1 integrating system. Results The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901–1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. Conclusion The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

scholarly journals Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage φFC1

2002 ◽  
Vol 184 (7) ◽  
pp. 1859-1864 ◽  
Author(s):  
Hee-Youn Yang ◽  
Young-Woo Kim ◽  
Hyo-Ihl Chang
Keyword(s):  
Pcr Amplification ◽  
Temperate Phage ◽  
Vector System ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Integration Vector ◽  
Site Specific Integration ◽  
A Site

ABSTRACT The genome of temperate phage φFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage φFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage φFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 × 103 transformants/μg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage φFC1 can be used for genetic engineering in E. faecalis and in other hosts.

scholarly journals Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

2009 ◽  
Vol 191 (6) ◽  
pp. 1933-1940 ◽  
Author(s):  
André Larouche ◽  
Paul H. Roy
Keyword(s):  
Antibiotic Resistance Genes ◽  
Cysteine Residue ◽  
Wild Type ◽  
Recombination Mechanism ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Environmental Bacterium ◽  
Target Sites ◽  
A Site

ABSTRACT Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.

scholarly journals OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system

SpringerPlus ◽  
2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Phornsiri Pechsrichuang ◽  
Chomphunuch Songsiriritthigul ◽  
Dietmar Haltrich ◽  
Sittiruk Roytrakul ◽  
Peenida Namvijtr ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Expression System ◽  
E Coli

Heterologous protein production using thePichia pastoris expression system

Yeast ◽  
2005 ◽  
Vol 22 (4) ◽  
pp. 249-270 ◽  
Author(s):  
Sue Macauley-Patrick ◽  
Mariana L. Fazenda ◽  
Brian McNeil ◽  
Linda M. Harvey
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Production ◽  
Pastoris Expression System

scholarly journals Site-Specific Integrative Elements of Rhizobiophage 16-3 Can Integrate into Proline tRNA (CGG) Genes in Different Bacterial Genera

2002 ◽  
Vol 184 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Szabolcs Semsey ◽  
Béla Blaha ◽  
Krisztián Köles ◽  
László Orosz ◽  
Péter P. Papp
Keyword(s):  
Rhizobium Meliloti ◽  
Trna Genes ◽  
Target Sequence ◽  
Integrase Gene ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Tyrosine Recombinases ◽  
Recombination System ◽  
Integrase Protein ◽  
Site Specific Recombination System

ABSTRACT The integrase protein of the Rhizobium meliloti 41 phage 16-3 has been classified as a member of the Int family of tyrosine recombinases. The site-specific recombination system of the phage belongs to the group in which the target site of integration (attB) is within a tRNA gene. Since tRNA genes are conserved, we expected that the target sequence of the site-specific recombination system of the 16-3 phage could occur in other species and integration could take place if the required putative host factors were also provided by the targeted cells. Here we report that a plasmid (pSEM167) carrying the attP element and the integrase gene (int) of the phage can integrate into the chromosomes of R. meliloti 1021 and eight other species. In all cases integration occurred at so-far-unidentified, putative proline tRNA (CGG) genes, indicating the possibility of their common origin. Multiple alignment of the sequences suggested that the location of the att core was different from that expected previously. The minimal attB was identified as a 23-bp sequence corresponding to the anticodon arm of the tRNA.

scholarly journals A self-inducible heterologous protein expression system in Escherichia coli

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
L. Briand ◽  
G. Marcion ◽  
A. Kriznik ◽  
J. M. Heydel ◽  
Y. Artur ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Expression System ◽  
Heterologous Protein Expression ◽  
Protein Expression System
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