Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

ABSTRACT We have developed a versatile Bacillus brevisexpression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.

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Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.42-49.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 42-49

Author(s):

K H Holt ◽

L Olson ◽

W S Moye-Rowley ◽

J E Pessin

Keyword(s):

Gene Fusion ◽

Kinase Activity ◽

Expression System ◽

Sh2 Domain ◽

Full Length ◽

Transactivation Domain ◽

Phosphatidylinositol 3 Kinase ◽

Sh2 Domains ◽

Amino Terminal ◽

Phosphatidylinositol 3

Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110.

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Identification of Dimeric Structure of Proteins by Use of the Glutathione S-Transferase-Fusion Expression System

Analytical Biochemistry ◽

10.1006/abio.1995.1300 ◽

1995 ◽

Vol 227 (2) ◽

pp. 396-399 ◽

Cited By ~ 8

Author(s):

T. Nemoto ◽

M. Ota ◽

Y. Oharanemoto ◽

M. Kaneko

Keyword(s):

Expression System ◽

Fusion Expression ◽

Glutathione S Transferase ◽

Dimeric Structure ◽

Structure Of Proteins

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Development of a Heterologous Gene Expression System for Use in Lactococcus Lactis

Recombinant Protein Production with Prokaryotic and Eukaryotic Cells. A Comparative View on Host Physiology ◽

10.1007/978-94-015-9749-4_19 ◽

2001 ◽

pp. 269-275 ◽

Cited By ~ 8

Author(s):

L. Bredmose ◽

S. M. Madsen ◽

A. Vrang ◽

P. Ravn ◽

M. G. Johnsen ◽

...

Keyword(s):

Gene Expression ◽

Lactococcus Lactis ◽

Heterologous Gene Expression ◽

Expression System ◽

Heterologous Gene ◽

Gene Expression System

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Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis

Canadian Journal of Microbiology ◽

10.1139/cjm-2019-0261 ◽

2020 ◽

Vol 66 (1) ◽

pp. 39-45

Author(s):

Marzieh Rezaei ◽

Mohammad Rabbani Khorasgani ◽

Sayyed Hamid Zarkesh Esfahani ◽

Rahman Emamzadeh ◽

Hamid Abtahi

Keyword(s):

Fusion Protein ◽

Lactococcus Lactis ◽

Fusion Gene ◽

Brucella Melitensis ◽

Expression System ◽

Interleukin 2 ◽

Cell Factory ◽

Promising Alternative ◽

Alternative Expression ◽

Restriction Sites

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

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