Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-µm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Inflammation primes the kidney for recovery by activating AZIN1 A-to-I editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and Adenosine Deaminase isoform switching. We found that A-to-I editing of Antizyme Inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

Inhibition of Importin- α -Mediated Nuclear Localization of Dendrin Attenuates Podocyte Loss and Glomerulosclerosis.

SIGNIFICANCE STATEMENT: Nuclear translocation of dendrin is observed in injured podocytes, but the mechanism and its consequence are unknown. In nephropathy mouse models, dendrin ablation attenuates proteinuria, podocyte loss, and glomerulosclerosis. The nuclear translocation of dendrin promotes c-Jun N -terminal kinase phosphorylation in podocytes, altering focal adhesion and enhancing cell detachment-induced apoptosis. We identified mediation of dendrin nuclear translocation by nuclear localization signal 1 (NLS1) sequence and adaptor protein importin- α . Inhibition of importin- α prevents nuclear translocation of dendrin, decreases podocyte loss, and attenuates glomerulosclerosis in nephropathy models. Thus, inhibiting importin- α -mediated nuclear translocation of dendrin is a potential strategy to halt podocyte loss and glomerulosclerosis. BACKGROUND: Nuclear translocation of dendrin is observed in the glomeruli in numerous human renal diseases, but the mechanism remains unknown. This study investigated that mechanism and its consequence in podocytes. METHODS: The effect of dendrin deficiency was studied in adriamycin (ADR) nephropathy model and membrane-associated guanylate kinase inverted 2 ( MAGI2 ) podocyte-specific knockout ( MAGI2 podKO) mice. The mechanism and the effect of nuclear translocation of dendrin were studied in podocytes overexpressing full-length dendrin and nuclear localization signal 1-deleted dendrin. Ivermectin was used to inhibit importin- α . RESULTS: Dendrin ablation reduced albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Dendrin deficiency also prolonged the lifespan of MAGI2 podKO mice. Nuclear dendrin promoted c-Jun N -terminal kinase phosphorylation that subsequently altered focal adhesion, reducing cell attachment and enhancing apoptosis in cultured podocytes. Classical bipartite nuclear localization signal sequence and importin- α mediate nuclear translocation of dendrin. The inhibition of importin- α / ß reduced dendrin nuclear translocation and apoptosis in vitro as well as albuminuria, podocyte loss, and glomerulosclerosis in ADR-induced nephropathy and MAGI2 podKO mice. Importin- α 3 colocalized with nuclear dendrin in the glomeruli of FSGS and IgA nephropathy patients. CONCLUSIONS: Nuclear translocation of dendrin promotes cell detachment-induced apoptosis in podocytes. Therefore, inhibiting importin- α -mediated dendrin nuclear translocation is a potential strategy to prevent podocyte loss and glomerulosclerosis.

IgA-dominant infection-related glomerulonephritis with NAPlr-positive tubulointerstitial nephritis.

Infection-related glomerulonephritis (IRGN) is one of the most common causes of acute kidney injury (AKI). Positive glomerular staining of the nephritis-associated plasmin receptor (NAPlr) has been reported as a useful biomarker of IRGN. Although the infection can provoke acute tubulointerstitial nephritis (AIN), there are few reports of positive staining for NAPlr with AIN. We report a case of methicillin-sensitive Staphylococcus aureus (MSSA) infection-related nephritis complicated with AIN, which showed positive staining for tubulointerstitial NAPlr. The patient developed AKI and nephrotic syndrome during an intraperitoneal MSSA infection. A diagnosis of IRGN complicated by infection-related acute tubulointerstitial nephritis (IRAIN) was made based on glomerular endocapillary proliferation with tubulointerstitial infiltrating cells and tubular atrophy. Tubulointerstitial infiltrating cells were positive for NAPlr staining and plasmin activity. Treatment of the infection by antibiotics and drainage did not improve the AKI, but steroid administration improved that. NAPlr evaluation is a helpful tool for identifying causes of AIN during infection.

Alport syndrome: A case study of chronic type A aortic dissection.

A 39-year-old woman with a history of Alport syndrome was admitted to our hospital for heart failure due to severe aortic regurgitation. Computed tomography revealed a chronic type A aortic dissection that required valve-sparing aortic root replacement. The pathological examination demonstrated that elastic fibers in the tunica media of the aortic wall are torn and severely disorganized. Immunostaining showed fragmented alpha 5 chains, indicating Alport syndrome. These findings imply Alport syndrome may have connective tissue vulnerability, rendering patients susceptible to the development of aortic disease at a young age.

Renal Hyperperfusion Injury After Percutaneous Angioplasty for Renovascular Hypertension as a Sequela of Neuroblastoma.

Hyperperfusion injury is a rare but critical complication associated with revascularization for long-standing severe artery stenosis. Here we report a rare case of a patient with renal hyperperfusion injury after undergoing percutaneous transluminal renal angioplasty for renovascular hypertension as a sequela of neuroblastoma after radiation therapy. (Level of Difficulty: Advanced.).

Suppressor of cytokine signalling 3 (SOCS3) expressed in podocytes attenuates glomerulonephritis and suppresses autoantibody production in an imiquimod-induced lupus model.

OBJECTIVE: Recently, podocytes have been recognised not only as a physical barrier to prevent urinary protein loss but also as producers of proinflammatory cytokines. However, the roles of podocytes in the pathogenesis of lupus nephritis (LN) remain largely unknown. This study aims to determine the roles of suppressor of cytokine signalling (SOCS) family members expressed in glomeruli in the regulation of LN. METHODS: We investigated the expression of SOCS family members in glomeruli in murine lupus model induced by repeated epicutaneous administration of the TLR7/8 agonist imiquimod. We also investigated the roles of SOCS3 expressed in podocytes in the imiquimod-induced glomerulonephritis and systemic autoimmunity by using podocyte-specific SOCS3-deficient mice (podocin-Cre x SOCS3fl/fl mice (SOCS3-cKO mice)). Finally, we investigated the expression of proinflammatory cytokines and chemokines in SOCS3-deficient podocyte cell lines. RESULTS: qPCR analysis revealed that among SOCS family members, SOCS3 was preferentially induced in glomeruli on epicutaneous administration of imiquimod and that interleukin 6 (IL-6) induced SOCS3 expression in podocyte cell lines. SOCS3-cKO mice exhibited severe glomerulonephritis, high levels of serum creatinine and urine albumin and decreased survival rate compared with control SOCS3-WT mice. Levels of anti-double-strand DNA antibody, SOCS (GC) formation and the numbers of follicular helper T (Tfh) cells and GC B cells in the spleen were higher in SOCS3-cKO mice than those in SOCS3-WT mice. Serum IL-6 levels and expression of IL-6 mRNA in glomeruli were also elevated in SOCS3-cKO mice. IL-6-induced IL-6 expression was enhanced in SOCS3-deficient podocyte cell lines compared with that in SOCS3-sufficient podocyte cell lines. CONCLUSION: SOCS3 expressed in podocytes plays protective roles for the development of glomerulonephritis and inhibits autoantibody production in the imiquimod-induced lupus model presumably by suppressing IL-6 production of podocytes.

Impairment of Proteasome Function in Podocytes Leads to CKD.

BACKGROUND: The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system (APLS) are major intracellular degradation procedures. The importance of the APLS in podocytes is established, but the role of the UPS is not well understood. METHODS: To investigate the role of the UPS in podocytes, mice were generated that had deletion of Rpt3 (Rpt3pdKO), which encodes an essential regulatory subunit required for construction of the 26S proteasome and its deubiquitinating function. RESULTS: Rpt3pdKO mice showed albuminuria and glomerulosclerosis, leading to CKD. Impairment of proteasome function caused accumulation of ubiquitinated proteins and of oxidative modified proteins, and it induced podocyte apoptosis. Although impairment of proteasome function normally induces autophagic activity, the number of autophagosomes was lower in podocytes of Rpt3pdKO mice than in control mice, suggesting the autophagic activity was suppressed in podocytes with impairment of proteasome function. In an in vitro study, antioxidant apocynin and autophagy activator rapamycin suppressed podocyte apoptosis induced by proteasome inhibition. Moreover, rapamycin ameliorated the glomerular injury in the Rpt3pdKO mice. The accumulation of ubiquitinated proteins and of oxidative modified proteins, which were detected in the podocytes of Rpt3pdKO mice, is a characteristic feature of aging. An aging marker was increased in the podocytes of Rpt3pdKO mice, suggesting that impairment of proteasome function promoted signs of aging in podocytes. CONCLUSIONS: Impairment of proteasome function in podocytes led to CKD, and antioxidants and autophagy activators can be therapeutic agents for age-dependent CKD.

MAGI-2 orchestrates the localization of backbone proteins in the slit diaphragm of podocytes.

Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained.

Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca2+ and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.

A rare case of nephrotic syndrome associated with Dent's disease: a case report.

Dent's disease is a rare X-linked condition caused by a mutation in CLCN5 and OCRL gene, which impair the megalin-cubilin receptor-mediated endocytosis in kidney's proximal tubules. Thus, it may manifest as nephrotic-range low-molecular-weight proteinuria (LMWP). On the other hand, glomerular proteinuria, hypoalbuminemia, and edema formation are the key features of nephrotic syndrome that rarely found in Dent's disease. Here, we reported a man in his 30 s with Dent's disease presented with leg edema for 5 days. The laboratory results revealed hypoalbuminemia and a decrease of urine ß2-microglobulin/urine protein ratio (Uß2-MG /UP), indicating that the primary origin of proteinuria shifted from LMWP to glomerular proteins. The kidney biopsy revealed glomerular abnormality and calcium deposition in the renal medulla. Electron microscopy of the kidney tissue indicated extensive foot-process effacement of the glomerular podocytes and degeneration of tubular epithelium. After a combination of treatment with prednisolone and cyclosporine (CyA), the nephrotic syndrome was remitted. Given the atypical clinical presentation and the shift of LMWP to glomerular proteinuria in this patient, glomerulopathy and the Dent's disease existed separately in this patient.

Glomerulosclerosis Induced by Deficiency of Membrane-Associated Guanylate Kinase Inverted 2 in Kidney Podocytes.

Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a component of the slit diaphragm (SD) of glomerular podocytes. Here, we investigated the podocyte-specific function of MAGI-2 using newly generated podocyte-specific MAGI-2-knockout (MAGI-2-KO) mice. Compared with podocytes from wild-type mice, podocytes from MAGI-2-KO mice exhibited SD disruption, morphologic abnormalities of foot processes, and podocyte apoptosis leading to podocyte loss. These pathologic changes manifested as massive albuminuria by 8 weeks of age and glomerulosclerosis and significantly higher plasma creatinine levels at 12 weeks of age; all MAGI-2-KO mice died by 20 weeks of age. Loss of MAGI-2 in podocytes associated with decreased expression and nuclear translocation of dendrin, which is also a component of the SD complex. Dendrin translocates from the SD to the nucleus of injured podocytes, promoting apoptosis. Our coimmunoprecipitation and in vitro reconstitution studies showed that dendrin is phosphorylated by Fyn and dephosphorylated by PTP1B, and that Fyn-induced phosphorylation prevents Nedd4-2-mediated ubiquitination of dendrin. Under physiologic conditions in vivo, phosphorylated dendrin localized at the SDs; in the absence of MAGI-2, dephosphorylated dendrin accumulated in the nucleus. Furthermore, induction of experimental GN in rats led to the downregulation of MAGI-2 expression and the nuclear accumulation of dendrin in podocytes. In summary, MAGI-2 and Fyn protect dendrin from Nedd4-2-mediated ubiquitination and from nuclear translocation, thereby maintaining the physiologic homeostasis of podocytes, and the lack of MAGI-2 in podocytes results in FSGS.

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.

Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA.

The development of integral membrane protein cell-free synthesis permits in-vitro labeling of accessible cysteines for real-time FRET and LRET measurements. The functional integrity of these synthetic ion channel proteins has been verified at the whole oocyte level by direct injection into, and recording from, Xenopus oocytes. However, the microscopic single-channel properties of cell-free translated protein have not been systematically examined. In the present study, we compare patch-clamp currents originating from cell-free protein with currents derived from mRNA injection, using the same (single-Cys) inward rectifier DNA template (C189-Kir1.1b). Results indicate that cell-free Kir protein, incorporated into liposomes and injected into oocytes, is trafficked to the plasma membrane where it inserts in an outside-out orientation and exhibits single-channel characteristics identical to that derived from a corresponding mRNA.

Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; and (6) the comparative performance of E. coli cells or wheat germ cell-free translation. Optimal promoter/repressor and fusion tag configurations for each expression system are discussed.

Automated cell-free protein production methods for structural studies.

In contrast to cell-based protein expression, cell-free production is highly consistent, scalable, and amenable to automation. Robots can handle many samples and perform repetitive procedures that are otherwise prone to human error. Here is described commercially available robotics for a wheat germ cell-free system with emphasis on practical applications for structural and functional studies. In addition, described is a cell-free method for preparing protein complexes.

Cell-free translation of biofuel enzymes.

In nature, bacteria and fungi are able to utilize recalcitrant plant materials by secreting a diverse set of enzymes. While genomic sequencing efforts offer exhaustive lists of genes annotated as potential polysaccharide-degrading enzymes, biochemical and functional characterizations of the encoded proteins are still needed to realize the full potential of this natural genomic diversity. This chapter outlines an application of wheat germ cell-free translation to the study of biofuel enzymes using genes from Clostridium thermocellum, a model cellulolytic organism. Since wheat germ extract lacks enzymatic activities that can hydrolyze insoluble polysaccharide substrates and is likewise devoid of enzymes that consume the soluble sugar products, the cell-free translation reactions provide a clean background for production and study of the reactions of biofuel enzymes. Examples of assays performed with individual enzymes or with small sets of enzymes obtained directly from cell-free translation are provided.

Cell-free protein synthesis for functional and structural studies.

Recent advances in cell-free protein expression systems have made them reliable and practical for functional and structural studies of a wide variety of proteins. In particular, wheat germ cell-free translation can consistently produce target proteins in microgram quantities from relatively inexpensive, small-scale reactions. Here we describe our small-scale protein expression method for rapidly producing proteins for functional assay and techniques for determining if the target is suitable for scale-up to amounts potentially needed for structure determination. The cell-free system is versatile and can be easily customized with the inclusion of additives. We describe simple modifications used for producing membrane proteins.