Membrane Protein Production in Lactococcus lactis for Functional Studies.

BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/Principal FindingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/SignificanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

3 - Production and Purification of Recombinant Membrane Proteins

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).

MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production

Automated production of functional membrane proteins using eukaryotic cell-free translation systems

RETURN TO ISSUEPREVNewsNEXTMISTIC BOOSTS PROTEIN YIELDSDiscovery allows eukaryotic membrane proteins to be made in high yieldsAMANDA YARNELLCite this: Chem. Eng. News 2005, 83, 9, 13Publication Date (Print):February 28, 2005Publication History Published online14 November 2010Published inissue 28 February 2005https://pubs.acs.org/doi/10.1021/cen-v083n009.p013https://doi.org/10.1021/cen-v083n009.p013newsACS PublicationsCopyright © 2005 American Chemical SocietyArticle Views8Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options SUBJECTS:Peptides and proteins Get e-Alerts

Despite the great importance of human membrane proteins involved in detoxification mechanisms, their wide use for biochemical approaches is still hampered by several technical difficulties considering eukaryotic protein expression in order to obtain the large amounts of protein required for functional and/or structural studies. Lactococcus lactis has emerged recently as an alternative heterologous expression system to Escherichia coli for proteins that are difficult to express. The aim of this work was to check its ability to express mammalian membrane proteins involved in liver detoxification, i.e., CYP3A4 and two isoforms of MGST1 (rat and human). Genes were cloned using two different strategies, i.e., classical or Gateway-compatible cloning, and we checked the possible influence of two affinity tags (6×-His-tag and Strep-tag II). Interestingly, all proteins could be successfully expressed in L. lactis at higher yields than those previously obtained for these proteins with classical expression systems (E. coli, Saccharomyces cerevisiae) or those of other eukaryotic membrane proteins expressed in L. lactis. In addition, rMGST1 was fairly active after expression in L. lactis. This study highlights L. lactis as an attractive system for efficient expression of mammalian detoxification membrane proteins at levels compatible with further functional and structural studies.

Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins, for various applications. This article details a series of protocols that progress towards the production of integral membrane proteins, their extraction and purification in the presence of detergents, and their eventual reconstitution in lipid nanoparticles. These basic procedures can be further optimized to provide integral membrane protein samples that are compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides general guidelines, technical hints, and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian proteins. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Production of membrane proteins in a P. pastoris recombinant clone using methanol induction Basic Protocol 2: Preparation of whole-membrane fractions Alternate Protocol 1: Preparation of yeast protoplasts Basic Protocol 3: Extraction of membrane proteins from whole-membrane fractions Basic Protocol 4: Purification of membrane proteins Alternate Protocol 2: Purification of membrane proteins from yeast protoplasts Alternate Protocol 3: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteins Basic Protocol 5: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles.

In the past decade, the methylotrophic yeast Pichia pastoris has proved to be one of the most efficient systems for mass production of recombinant eukaryotic membrane proteins (MPs), leading to the crystallization and structure determination for a variety of them. The actual overexpression of functional MPs achieved with this system is, however, often accompanied by the formation of a variable but significant proportion of misfolded and/or aggregated proteins that are co-extracted and co-purified during the purification process. In order to minimize this unwanted phenomenon, we devised a novel procedure in which MPs produced in Pichia pastoris are directly solubilized from whole cells instead of crude membrane preparation. This approach aims at favoring the extraction of correctly folded membrane proteins that have been targeted to the plasma membrane, limiting the solubilization of the misfolded proteins and protein aggregates that are stored in internal membrane compartments. The method described herewith is based on the formation of protoplasts through enzymatic treatment prior to protein solubilization. This chapter details a set of protocols going from yeast cell preparation and protein solubilization to purification using affinity and size exclusion chromatography.

Preface INTRODUCTION Expression Solubilization and Structural Methods Abbreviations PART I: Expression Systems BACTERIAL SYSTEMS Introduction Understanding the Problem Vector/Promoter Types T7 Expression System Tunable T7 Expression Systems Other Useful Membrane Protein Expression Strains Clone Stability Media Types Fusion Partners/Membrane Targeting Peptides Chaperone Overexpression Cautionary Notes Related to Chaperone Overexpression Emerging Role of Quality Control Proteases Tag Selection Potential Expression Yield Strategies to Overcome Protein Instability MEMBRANE PROTEIN EXPRESSION IN SACCHAROMYCES CEREVISIAE Introduction Getting Started Special Considerations Case Studies Conclusions EXPRESSION SYSTEMS: PICHIA PASTORIS Introduction A (Brief) Summary on the (Long) History of P. pastoris Introducing P. pastoris as a Biotechnological Tool: Its (Extended) Strengths and (Limited) Weaknesses Basics of the P. pastoris Expression System Successful Large-Scale Expression of Membrane Proteins Using P. pastoris Guidelines for Optimizing Membrane Protein Expression in P. pastoris Using GPCRs as Models Conclusions and Future Directions HETEROLOGOUS PRODUCTION OF ACTIVE MAMMALIAN G-PROTEIN-COUPLED RECEPTORS USING BACULOVIRUS-INFECTED INSECT CELLS Introduction Experimental Conclusions and Future Perspectives MEMBRANE PROTEIN EXPRESSION IN MAMMALIAN CELLS Introduction Mammalian Systems Case Studies Conclusions MEMBRANE PROTEIN PRODUCTING USING PHOTOSYNTHETIC BACTERIA: A PRACTICAL GUIDE Introduction Preparation of Expression Constructs Transfer of Plasmid DNA to Rhodobacter via Conjugal Mating Small-Scale Screening for Expression and Localization of Target Protein in Rhodobacter Large-Scale Culture Detergent Solubilization and Chromatographic Purification of Expressed Membrane Proteins Protein Identification and Assessment of Purity Preparations of Specialized Rhodobacter Membranes Appendix: Media and Buffer Formulations PART II: Protein-Specific Considerations PERIPHERAL MEMBRANE PROTEIN PRODUCTION FOR STRUCTURAL AND FUNCTIONAL STUDIES Introduction Case Studies of Peripheral Membrane Proteins Conclusions EXPRESSION OF G-PROTEIN-COUPLED RECEPTORS Introduction Bacterial Expression of GPCRs Expression of GPCrs in Inclusion Bodies and Refolding Expression of GPCrs in Yeast Expression of GPCrs in Insect Cells Expression of GPCrs in Mammalian Cell Lines Expression of GPCrs in Retina Rod Cells Expression of GPCrs in a Cell-Free System Stabilization of GPCrs during Solubilization and Purification Conclusions STRUCTURAL BIOLOGY OF MEMBRANE PROTEINS Introduction Folding and Structural Analysis of Membrane Proteins Test Cases PART III: Emerging Methods and Approaches ENGINEERING INTEGRAL MEMBRANE PROTEINS FOR EXPRESSION AND STABILITY Introduction Engineering Higher Expression Engineering Higher Stability Conclusions EXPRESSION AND PURIFICATION OF G-PROTEIN-COUPLED RECEPTORS FOR NUCLEAR MAGNETIC RESONANCE STRUCTURAL STUDIES Introduction: G-Protein-Coupled Receptor Superfamily CXCR1 GPCR Structures NMR Studies of GPCRs Expression Systems Cloning of CXCR1 into pGEX2a Expression of CXCR1 Purification Refolding and Reconstitution Binding and Activity Measurements NMR Spectra SOLUBILIZATION, PURIFICATION, AND CHARACTERIZATION OF INTEGRAL MEMBRANE PROTEINS Introduction Solubilization of IMPs IMP Purification Characterization of Solubilized IMPs STABILIZING MEMBRANE PROTEINS IN DETERGENT AND LIPID SYSTEMS Introduction Choice of Detergent: Solubilization versus Stability Mitigating Protein Denaturation Making or Selecting a Stable Protein Conclusions RAPID OPTIMIZATION OF MEMBRANE PROTEIN PRODUCTION USING GREEN FLUORESCENT PROTEIN-FUSIONS AND LEMO21(DE3) Introduction Main Protocol Materials Expression and Isolation of GFP-His8 Conclusions

We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.

Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio-function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta- barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

In the past, dexamethasone and glucagon has been shown to have a variable effect on the expression of soluble liver enzymes during development. The aim of the present study was to investigate the effect of dexamethasone and glucagon on the expression of plasma membrane proteins during ontogeny. Our previous studies have demonstrated a significant increase of the asialoglycoprotein receptor (ASGR) and 110,000 Mr glycoprotein at birth, decrease in alkaline phosphatase (AP) and gammaglutamyltranspeptidase (GT) and very little change in leucine aminopeptidase (LAP) after birth. Buffalo rat fetuses at 18 days of gestation and 1-day-old newborns were injected (single and daily, respectively) with dexamethasone (2 and 4 μg, respectively) or glucagon (25 and 50 μg, respectively). Fetuses were sacrificed at 22 days of gestation and newborns at 3, 5, and 7 days of age. Total post-nuclei membrane vesicles were prepared from rat livers using a sucrose gradient method. Quantitation of levels of membrane proteins was performed using immunological and/or enzymatic methods. In fetal rats, dexamethasone did not affect the expression of membrane proteins. In contrast, glucagon increased significantly the levels of AP, LAP, and GT. In newborns, glucagon did not alter membrane protein levels. However, dexamethasone dramatically increased six-fold the level of GT while the amounts of ASGR, AP, and LAP were reduced by two-to four-fold. These data indicate that there is a differential effect of dexamethasone and glucagon on the expression of membrane proteins during late intrauterine and early extrauterine stages of development.

For acquisition of iron, an essential nutrient, most microorganisms produce siderophores (low-molecular-weight iron-chelating compounds) and membrane proteins to serve as receptors for the iron-siderophore complexes. The gonococcus does not appear to produce a siderophore, since the quantity of siderophore detected by bioassays of culture supernatants from strains F62 and FA19 was never greater than the amount present in the uninoculated medium. Iron limitation of the laboratory strains F62 and FA19 and 12 recent clinical isolates resulted in the expression of several iron-repressible membrane proteins. The expression of proteins in the apparent molecular weight range of 70,000 to 100,000 was strain dependent. All strains expressed 36,000-dalton (36K) and 19.5K proteins. FA19 and F62 were also grown in medium containing iron sources commonly encountered in vivo (i.e., transferrin, lactoferrin, hemoglobin, or hemin). Comparison of growth rates indicates that transferrin and lactoferrin were more readily utilized as iron sources than hemin and hemoglobin were. Expression of the iron-repressible proteins varied depending upon the iron source. Fewer iron-repressible proteins were observed when cells were supplied with transferrin or lactoferrin than when the cultures were grown with either hemin or hemoglobin. The 36K protein was expressed with all four iron sources.