Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins

Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included.     

Rat liver 10-formyltetrahydrofolate dehydrogenase, carbamoyl phosphate synthetase 1 and betaine homocysteine S-methytransferase were co-purified on Kunitz-type soybean trypsin inhibitor-coupled sepharose CL-4B

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.     

植物甲酸脱氢酶基因的表达调控及其生理功能研究进展

甲酸脱氢酶（FDH）是植物中普遍存在的一种含量丰富的酶。甲酸脱氢酶也是一种NAD依赖的酶,它催化甲酸氧化成二氧化碳的可逆反应。植物FDH是植物一碳代谢的一部分,它在植物响应各种环境胁迫、低氧或缺氧过程中发挥着重要的作用,因此在农学生产上具有很大的应用潜力。最近植物来源的FDH基因克隆和表达调控及其生理学功能等各方面的研究都取得很多重要的进展。综述了近年来植物来源的FDH基因克隆和表达调控及其生理学功能方面的研究进展。     

Cloning, nucleotide sequencing, and expression in Escherichia coli of the gene for formate dehydrogenase of Paracoccus sp. 12-A, a formate-assimilating bacterium

The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50 degrees C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.     

Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.     

甲醛氧化途径关键酶重组蛋白的生化特性及固定化酶吸收甲醛效果研究

甲醛脱氢酶(formaldehyde dehydrogenase,ADH)与甲酸脱氢酶(formate dehydrogenase,FDH)是甲醛氧化途径的两个关键酶.恶臭假单胞菌(Pseudomonas putida)的PADH是一种不依赖谷胱甘肽可以把游离甲醛直接氧化为甲酸的脱氢酶,博伊丁假丝酵母菌(Candida boidinii)的FDH在有NAD+存在时可以把甲酸氧化为二氧化碳.以基因组DNA为模板用PCR方法,从P.putida中扩增出PADH基因的编码区(padh),从C.boidinii中扩增出FDH的编码区(fdh),然后亚克隆到pET-28a(+)中分别构建这两个基因的原核表达载体pET-28a-padh和pET-28a-fdh,转化大肠杆菌,利用IPTG诱导重组蛋白PADH和FDH的表达.通过优化条件使重组蛋白的表达量占菌体总蛋白的70%以上,通过亲和层析法纯化出可溶性PADH和FDH重组蛋白.对重组蛋白的生化特性分析结果表明:PADH在最适反应温度50℃的活性为1.95 U/mg;FDH在最适反应温度40℃的活性为0.376 U/mg.所表达的重组蛋白与之前报道过的相比,具有更好的热稳定性和更广的温度适应范围.将PADH、FDH两个重组蛋白及辅因子NAD+固定到聚丙烯酰胺载体基质上,对固定化酶甲醛吸收效果的初步分析结果显示固定化酶对空气中的甲醛有一定的吸收效果,说明这两种酶被固定后具有开发成治理甲醛污染环保产品的潜力.     

ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase

Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO₂ in an NADP⁺-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO₂ production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.     

竹芋科和蕨类室内盆栽植物对甲醛净化作用的研究

以竹芋科和蕨类室内盆栽植物各10种为试验材料,分别置于体积为1.0 m×1.0m×0.8m密封玻璃箱内,甲醛起始浓度均设置为15 mg/m3,连续观察7d.结果表明:卷叶巢蕨(Neottopteris nidus cv.Volulum)、矩叶肖竹芋(Calathea lubbersiana)对甲醛抗性最强(Ⅰ级);银线竹芋(C.ornata)、银羽斑竹芋(C.setosa)、翠叶竹芋(C.freddy)和彩虹竹芋(C.roseo-picta)抗性较强(Ⅱ级);巢蕨(N.nidus)、傅氏凤尾蕨(Pteris fauriei)、银脉凤尾蕨(P.ensi formis cv.Victoriae)、银心大叶凤尾蕨(P.cretica cv.Albolineata)、肾蕨(Nephrolepis cordifolia)、华南毛蕨(Cyclosorus parasiticus)、乌毛蕨(Blechnum orientale)、花叶竹芋(Maranta bicolor)和天鹅绒竹芋(C.zebrina)抗性最差(Ⅳ级).甲醛处理后,密封玻璃箱内甲醛浓度均呈递减变化,递减最快都集中在试验后1～3 d之间.吸收甲醛最快的植物是天鹅绒竹芋和星蕨(Microsorum punctatum),最慢的是华南毛蕨、银脉凤尾蕨、卷叶巢蕨和银羽斑竹芋.对甲醛处理产生伤害反应少或较少,而吸收能力强的前8种植物是:巢蕨、青苹果竹芋(C.rotundfolia)、银心大叶凤尾蕨、银线竹芋、二歧鹿角蕨(Platycerium bifurcatum)、卷叶巢蕨、彩虹竹芋和翠叶竹芋,可作为甲醛净化专用植物应用推广.     

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.     

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Characterization of haem and iron-sulphur centres by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy.

The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.     

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.     

Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.

The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.     

Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles

Rapid immobilization with the one-pot purification of galactitol dehydrogenase (GatDH) and formate dehydrogenase (FDH) is achieved by using iminodiacetic acid (IDA) with chelated Co²⁺ modified magnetic nanoparticles as a carrier. Lactate dehydrogenase (LDH) from recombinant Escherichia coli and FDH commencing Candida methylica were used as an auxiliary enzyme for the regeneration of NADH/NAD⁺ with a representative synthesis of (S)-1,2-propanediol and l-tagatose starting from hydroxyacetone and galactitol. The affinity magnetic nanoparticles were characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), while the purity of GatDH and FDH was assayed by SDS-PAGE analysis. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, showed optimal activity at pH 7 and 8 for (S)-1,2-propanediol and l-tagatose production, respectively. The immobilized enzyme system retained up to 70% of its activity after one week of repeated use. The use of affinity magnetic nanoparticles offers the advantage of a one-pot purification of His(6)-tagged GatDH and FDH followed by the production of rare sugar and chiral diol.     

Biomimetic-dye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron(R) Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix((R)) Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel((R)) A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4 degrees C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( K(D)) of the complex between immobilized BM6 and FDH was found to equal 0.05 muM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30 degrees C). (c) 1995 John Wiley & Sons, Inc.     

Physiological and quantitative proteomic analyses unraveling potassium deficiency stress response in alligator weed (<Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> L.) root

Key message

KEG is involved in mediating the proteasome-dependent degradation of FDH, a stress-responsive enzyme. The UPS may function to suppress FDH mediated stress responses under favorable growth conditions.

Abstract

Formate dehydrogenase (FDH) has been studied in bacteria and yeasts for the purpose of industrial application of NADH co-factor regeneration. In plants, FDH is regarded as a universal stress protein involved in responses to various abiotic and biotic stresses. Here we show that FDH abundance is regulated by the ubiquitin proteasome system (UPS). FDH is ubiquitinated in planta and degraded by the 26S proteasome. Interaction assays identified FDH as a potential substrate for the RING-type ubiquitin ligase Keep on Going (KEG). KEG is capable of attaching ubiquitin to FDH in in vitro assays and the turnover of FDH was increased when co-expressed with a functional KEG in planta, suggesting that KEG contributes to FDH degradation. Consistent with a role in regulating FDH abundance, transgenic plants overexpressing KEG were more sensitive to the inhibitory effects of formate. In addition, FDH is a phosphoprotein and dephosphorylation was found to increase the stability of FDH in degradation assays. Based on results from this and previous studies, we propose a model where KEG mediates the ubiquitination and subsequent degradation of phosphorylated FDH and, in response to unfavourable growth conditions, reduction in FDH phosphorylation levels may prohibit turnover allowing the stabilized FDH to facilitate stress responses.

     

Improved purification of Candida boidinii formate dehydrogenase

Formate dehydrogenase (FDH, EC 1.2.1.2) from Candida boidinii was purified to homogeneity. The two step procedure comprised anion exchange chromatography (2.9-fold purification, 85% step yield, elution with 35 mM KCl), followed by dye-ligand affinity chromatography on immobilized Cibacron Blue 3GA (1.4-fold purification, 75% step yield, elution with 0.15 mM NAD⁺/2 mM Na₂SO₃). The procedure afforded FDH at 63.8% overall yield and a specific activity of 7.2 units/mg. The purity of the final FDH preparation was evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), high performance gel filtration liquid chromatography (gfHPLC) and N-terminal amino acid sequencing. The analytical techniques showed the presence of a single polypeptide chain that corresponds to the molecular weight of 41 kDa (as determined by SDS-PAGE) and 81 kDa (as determined by gfHPLC).