Membrane Protein Expression in Lactococcus lactis

King, Martin S.; Boes, Christoph; Kunji, Edmund R. S.

doi:10.1016/bs.mie.2014.12.009

Cited by 24 publications

(17 citation statements)

References 29 publications

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“…For optimal expression in Lactococcus lactis [27] , residues 2–14 and 309–315 were removed by PCR, including the His-tag, and the resulting genes were cloned into the expression vector pNZ8048 under the control of a nisin A-inducible promoter [28] . The plasmids were transformed in L. lactis strain NZ9000 by electroporation, as previously described [27] , [29] , [30] . Vectors were isolated by miniprep (Qiagen), according to the manufacturer's instructions with one alteration: 10 mg ml -1 lysozyme was added to the lysis buffer and the resuspended cells were incubated at 55 °C for twenty minutes prior to lysis.…”

Section: Methodsmentioning

confidence: 99%

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

King

Kerr

Crichton

et al. 2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway.

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Section: Methodsmentioning

confidence: 99%

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

King

Kerr

Crichton

et al. 2016

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…The SLC25A4 gene was cloned into the L. lactis expression vector pNZ8048 by established procedures, 9 and the p.Lys33Gln variant was introduced and confirmed by sequencing. Growth of L. lactis , membrane isolation, vesicle preparation, transport assays, and western blot analysis were performed as reported.…”

Section: Methodsmentioning

confidence: 99%

“…Growth of L. lactis , membrane isolation, vesicle preparation, transport assays, and western blot analysis were performed as reported. 4 , 9 …”

Section: Methodsmentioning

confidence: 99%

Expanding the phenotype of de novo SLC25A4 -linked mitochondrial disease to include mild myopathy

et al. 2018

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ObjectiveTo determine the disease relevance of a novel de novo dominant variant in the SLC25A4 gene, encoding the muscle mitochondrial adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier, identified in a child presenting with a previously unreported phenotype of mild childhood-onset myopathy.MethodsImmunohistochemical and western blot analysis of the patient's muscle tissue were used to assay for the evidence of mitochondrial myopathy and for complex I–V protein levels. To determine the effect of a putative pathogenic p.Lys33Gln variant on ADP/ATP transport, the mutant protein was expressed in Lactococcus lactis and its transport activity was assessed with fused membrane vesicles.ResultsOur data demonstrate that the heterozygous c.97A>T (p.Lys33Gln) SLC25A4 variant is associated with classic muscle biopsy findings of mitochondrial myopathy (cytochrome c oxidase [COX]-deficient and ragged blue fibers), significantly impaired ADP/ATP transport in Lactococcus lactis and decreased complex I, III, and IV protein levels in patient's skeletal muscle. Nonetheless, the expression levels of the total ADP/ATP carrier (AAC) content in the muscle biopsy was largely unaffected.ConclusionsThis report further expands the clinical phenotype of de novo dominant SLC25A4 mutations to a childhood-onset, mild skeletal myopathy, without evidence of previously reported clinical features associated with SLC25A4-associated disease, such as cardiomyopathy, encephalopathy or ophthalmoplegia. The most likely reason for the milder disease phenotype is that the overall AAC expression levels were not affected, meaning that expression of the wild-type allele and other isoforms may in part have compensated for the impaired mutant variant.

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“…Because almost nothing is known about these nanostructures, the aim of this study was the isolation and detailed characterization of the main morphometric and physico-chemical properties of such a new class of protein deposits. For that, we selected L. lactis, the most used LAB in the field of recombinant protein production as cell factory [20,21]. Three relevant proteins in human and veterinary medicine such as bovine metalloproteinase 9 (MMP-9) and 2 (MMP-2) and interferon gamma (IFN-c) were used herein as model proteins.…”

Section: Introductionmentioning

confidence: 99%

Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology

et al. 2016

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Inclusion bodies (IBs) are protein-based nanoparticles formed in Escherichia coli through stereospecific aggregation processes during the overexpression of recombinant proteins. In the last years, it has been shown that IBs can be used as nanostructured biomaterials to stimulate mammalian cell attachment, proliferation, and differentiation. In addition, these nanoparticles have also been explored as natural delivery systems for protein replacement therapies. Although the production of these protein-based nanomaterials in E. coli is economically viable, important safety concerns related to the presence of endotoxins in the products derived from this microorganism need to be addressed. Lactic acid bacteria (LAB) are a group of food-grade microorganisms that have been classified as safe by biologically regulatory agencies. In this context, we have demonstrated herein, for the first time, the production of fully functional, IB-like protein nanoparticles in LAB. These nanoparticles have been fully characterized using a wide range of techniques, including field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, zymography, cytometry, confocal microscopy, and wettability and cell coverage measurements. Our results allow us to conclude that these materials share the main physico-chemical characteristics with IBs from E. coli and moreover are devoid of any harmful endotoxin contaminant. These findings reveal a new platform for the production of protein-based safe products with high pharmaceutical interest.Peer ReviewedPostprint (published version

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Membrane Protein Expression in Lactococcus lactis

Cited by 24 publications

References 29 publications

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier

Expanding the phenotype of de novo SLC25A4 -linked mitochondrial disease to include mild myopathy

Functional protein-based nanomaterial produced in microorganisms recognized as safe: A new platform for biotechnology

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