Membrane Protein Production in Lactococcus lactis for Structural Studies

Martens, Chloé

doi:10.1007/978-1-0716-0373-4_3

Cited by 3 publications

(3 citation statements)

References 30 publications

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“…This opens up the road to the elucidation of other MP structures in the future since the expression levels obtained for almost all the proteins is close to 1–2% and higher ( Table 2 , Table 3 and Table 4 ). Furthermore, the ability to label the MPs with SelenoMet resolves the diffraction data [ 137 ] and the availability of specific protocols developed for this purpose [ 138 ].…”

Section: Structures Resolved From Mps Expressed In L Lactismentioning

confidence: 99%

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Frelet-Barrand

2022

Biomolecules

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Membrane proteins play key roles in most crucial cellular processes, ranging from cell-to-cell communication to signaling processes. Despite recent improvements, the expression of functionally folded membrane proteins in sufficient amounts for functional and structural characterization remains a challenge. Indeed, it is still difficult to predict whether a protein can be overproduced in a functional state in some expression system(s), though studies of high-throughput screens have been published in recent years. Prokaryotic expression systems present several advantages over eukaryotic ones. Among them, Lactococcus lactis (L. lactis) has emerged in the last two decades as a good alternative expression system to E. coli. The purpose of this chapter is to describe L. lactis and its tightly inducible system, NICE, for the effective expression of membrane proteins from both prokaryotic and eukaryotic origins.

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Section: Structures Resolved From Mps Expressed In L Lactismentioning

confidence: 99%

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Frelet-Barrand

2022

Biomolecules

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“…Despite the advantages of this technique, there are only few examples of structural studies on membrane proteins using this approach, none of which ranges to the SLC superfamily [ 183 , 184 , 185 ]. Several examples of human SLC transporters expressed in prokaryotic expression hosts, purified in strong denaturing conditions, and on-column refolded are reported [ 23 , 186 , 187 , 188 ]. However, the isolation of a target protein from yeast membranes could be advantageous with respect to its recovery from inclusion bodies obtained following bacterial expression.…”

Section: Current Methodology For Purification and Functional Studies ...mentioning

confidence: 99%

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

Pochini

Galluccio

2022

Life

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For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets. Despite their critical role in human health, a large part of SLCs’ is ‘orphans’ for substrate specificity or function. Moreover, very few data are available concerning their 3D structure. On the basis of the human health benefits of filling these knowledge gaps, an understanding of protein expression in systems that allow functional production of these proteins is essential. Among the 500 known yeast species, S. cerevisiae and P. pastoris represent those most employed for this purpose. This review aims to provide a comprehensive state-of-the-art on the attempts of human SLC expression performed by exploiting yeast. The collected data will hopefully be useful for guiding new attempts in SLCs expression with the aim to reveal new fundamental data that could lead to potential effects on human health.

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“…The bacteriophage T7 promotor, often used for MP expression, can lead to overloading of the bacterial quality control system and accumulation of MPs in inclusion bodies [15,16]. Mitigation efforts drove the development of alternative bacterial expression hosts [15,17,18] and several E. coli strains temper the effects of the T7 promoter [19,20]. Other strains improve the expression levels of specific MPs [21] but MP functionality depends upon the strain [22].…”

Section: Choosing An Expression Systemmentioning

confidence: 99%

The high-throughput production of membrane proteins

Birch

Quigley

2021

Emerging Topics in Life Sciences

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Membrane proteins, found at the junctions between the outside world and the inner workings of the cell, play important roles in human disease and are used as biosensors. More than half of all therapeutics directly affect membrane protein function while nanopores enable DNA sequencing. The structural and functional characterisation of membrane proteins is therefore crucial. However, low levels of naturally abundant protein and the hydrophobic nature of membrane proteins makes production difficult. To maximise success, high-throughput strategies were developed that rely upon simple screens to identify successful constructs and rapidly exclude those unlikely to work. Parameters that affect production such as expression host, membrane protein origin, expression vector, fusion-tags, encapsulation reagent and solvent composition are screened in parallel. In this way, constructs with divergent requirements can be produced for a variety of structural applications. As structural techniques advance, sample requirements will change. Single-particle cryo-electron microscopy requires less protein than crystallography and as cryo-electron tomography and time-resolved serial crystallography are developed new sample production requirements will evolve. Here we discuss different methods used for the high-throughput production of membrane proteins for structural biology.

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Membrane Protein Production in Lactococcus lactis for Structural Studies

Cited by 3 publications

References 30 publications

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Lactococcus lactis, an Attractive Cell Factory for the Expression of Functional Membrane Proteins

Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

The high-throughput production of membrane proteins

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