Biotin-ubiquitin tagging of mammalian proteins in Escherichia coli

Ubiquitin has been used in protein expression for enhancing yields and biological activities of recombinant proteins. Biotin binds tightly and specifically to avidin and has been widely utilized as a tag for protein purification and monitoring. Here, we report a versatile system that takes the advantages of both biotin and ubiquitin for protein expression, purification, and monitoring. The tripartite system contained coding sequences for a leader biotinylation peptide, ubiquitin, and biotin holoenzyme synthetase in two reading frames under the control of T7 promoter. The expression and purification of several large mammalian enzymes as biotin-ubiquitin fusions were accomplished including human ubiquitin activating enzyme, SUMO activating enzymes, and aspartyl-tRNA synthetase. Expressed proteins were purified by one-step affinity column chromatography on monomeric avidin columns and purified proteins exhibited active function. Additionally, the ubiquitin protein hydrolase UBP41, expressed and purified as biotin-UBP41, efficiently and specifically cleaved off the biotin-ubiquitin tag from biotin-ubiquitin fusions to produce unmodified proteins. The present expression system should be useful for the expression, purification, and functional characterization of mammalian proteins and the construction of protein microarrays.     

Expression and purification of histidine-tagged proteins from the gram-positive Streptococcus gordonii SPEX system

Streptococcus gordonii (S. gordonii) has been used as a gram-positive bacterial expression vector for secreted or surface-anchored recombinant proteins. Fusion of the gram-positive bacterial N-terminal signal sequence to the target protein is all that is required for efficient export. This system is termed SPEX for Surface Protein EXpression and has been used to express proteins for a variety of uses. In this study, the SPEX system has been further developed by the construction of vectors that express polyhistidine-tagged fusion proteins. SPEX vectors were constructed with an N-terminal or C-terminal histidine tag. The C-repeat region (CRR) from Streptococcus pyogenes M6 protein and the Staphylococcus aureus nuclease A (NucA) enzyme were tested for expression. The fusion proteins were purified using metal affinity chromatography (MAC). Results show that the fusion proteins were expressed and secreted from S. gordonii with the His tag at either the N- or C-terminal position and could be purified using MAC. The M6 fusions retained immunoreactivity after expression and purification as determined by immunoblots and ELISA analyses. In addition, NucA fusions retained functional activity after MAC purification. The M6-His and NucA-His fusions were purified approximately 15- and 10-fold respectively with approximately 30% recovery of protein using MAC. This study shows that the polyhistidine tag in either the N- or C-terminal position is a viable way to purify secreted heterologous proteins from the supernatant of recombinant S. gordonii cultures. This study further illustrates the value of the SPEX system for secreted expression and purification of proteins.     

Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli

Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.     

Purification of a ubiquitin protein peptidase from yeast with efficient in vitro assays

In eukaryotic cells ubiquitin is synthesized as a polyubiquitin protein or as a protein fused at the carboxyl terminus to other polypeptides. An enzyme activity, ubiquitin protein peptidase, has been proposed to process these precursors by cleaving the peptide bond between adjoining ubiquitin molecules or between ubiquitin and the fused peptides. Using the cleavage of a 35S-labeled yeast ubiquitin protein fused to a synthetic 38-residue peptide obtained by in vivo metabolic labeling in Escherichia coli in an expression system based on the interaction of bacteriophage T7 RNA polymerase and its promoter, it is possible to detect a processing activity in soluble yeast extract. The specificity of the cleavage suggests this activity could be the in vivo processing activity for various ubiquitin precursor proteins in yeast cells. A similarly labeled ubiquitin protein fused to one cysteine residue was also utilized to detect an activity capable of removing a single cysteine residue from ubiquitin in a soluble extract. Employing assays based on the cleavage of labeled ubiquitin protein fusions, a ubiquitin protein peptidase activity from Saccharomyces cerevisiae was purified about 15,000-fold to yield a protein mixture consisting of only a few protein species. The major protein band which comigrated with the activities in in vitro assays has an apparent molecular weight of 29,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two other protein species, about 20,000 and 10,000 in molecular weight, also comigrated with the in vitro activities throughout the purification procedure. Though our most purified protein fraction was shown to cleave various artificial ubiquitin protein fusions under our experimental conditions, it cannot cleave a ubiquitin dimer protein, suggesting the existence of functionally distinct ubiquitin protein peptidases. Our experimental protocol for preparing various labeled ubiquitin protein precursors provides a means to explore various processing enzymes existing in cells. The same protocol may also be adapted to prepare substrates for the study of other specific protein processing enzymes.     

The macrophage-induced gene (mig) of Mycobacterium avium encodes a medium-chain acyl-coenzyme A synthetase.

The macrophage-induced gene (mig) of Mycobacterium avium has been associated with virulence, but the functions of the gene product were still unknown. Here we have characterized the Mig protein by biochemical methods. A plasmid with a histidine-tagged fusion protein was constructed for expression in Escherichia coli. Mig was detected as a 60 kDa protein after expression and purification of the recombinant gene product. The sequence of the fusion gene and of the parent gene in M. avium were reexamined. This confirmed that the mig gene encodes a 550 amino acid protein (58 kDa) instead of a 295 amino acid protein (30 kDa) as predicted before. The 550 amino acid Mig exhibits a high degree of homology to bacterial acyl-CoA synthetases. Two artificial 30 kDa derivatives of Mig were expressed and purified as histidine-tagged fusion proteins in E. coli. These proteins and the 58.6 kDa histidine-tagged Mig protein were analysed for activity with an acyl-CoA synthetase assay. Among the three investigated proteins, only the 58.6 kDa Mig exhibited detectable activity as an acyl-CoA synthetase (EC 6.2.1.3) with saturated medium-chain fatty acids, unsaturated long-chain fatty acid and some aromatic carbon acids as substrates. Enzymatic activity could be inhibited by 2-hydroxydodecanoic acid, a typical inhibitor of medium-chain acyl-CoA synthetases. We postulate a novel medium-chain acyl-CoA synthetase motif. We have investigated the biochemical properties of Mig and suggest that this enzyme is involved in the metabolism of fatty acid during mycobacterial survival in macrophages.     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins

In order to study 1) the mechanisms responsible for generating free ubiquitin monomer and 2) the function of ubiquitin carboxyl extension proteins in eukaryotes, we have developed a system for expression of human ubiquitin carboxyl extension proteins in prokaryotic and eukaryotic hosts. When expressed in Saccharomyces cerevisiae, the intact ubiquitin carboxyl extension proteins were rapidly processed to free ubiquitin monomer and extension protein. Furthermore, expression in this host conferred a slow growth phenotype mediated by the extension protein. Expression in Escherichia coli did not result in processing of the fusion proteins. However, when the expressed fusion proteins were purified from E. coli and incubated with a rabbit reticulocyte extract, the proteins were rapidly processed to free ubiquitin monomer and extension protein. These results show that human ubiquitin carboxyl extension proteins are processed to ubiquitin and extension protein when expressed in eukaryotic but not prokaryotic cells and that pre- and co-translational events are not necessary for their processing. Establishment of this system will allow for large scale purification of these proteins which will aid future studies on the function and structure of ubiquitin carboxyl extension proteins, as well as the mechanisms responsible for their processing.     

人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。     

Harnessing synthetic biology to enhance heterologous protein expression

The ability to express heterologous proteins in microbial hosts is crucial for many areas of research and technology. In most cases, however, successful expression and purification of the desired protein require fusion to another protein. To date, all fusion partners have been chosen from natural sequences, which evolved for other purposes, and may not be optimal fusion partners. However, the rise of synthetic biology and protein design make it possible to design and optimize fusion proteins using novel sequences that did not arise in nature. Here, we describe a series of De novo Expression Enhancer Proteins (DEEPs) that facilitate high‐level expression and facile purification of heterologous proteins and peptides. To test the DEEP system, a de novo protein was fused to several target proteins covering a range of sizes and solubilities. In all cases, fusions to DEEP outperformed fusions to SUMO, a commonly used natural fusion partner. The availability of novel proteins that can be engineered for specific fusion applications could be beneficial to enhance the expression of a wide range of heterologous proteins.     

Dihydrofolate reductase as a new "affinity handle".

Dihydrofolate reductase (DHFR) has been demonstrated to be a versatile "affinity handle" for expression of recombinant proteins. The DHFR "handle" has advantages not only in terms of efficiency of expressing the fusion protein as a soluble form but also in stabilizing unstable polypeptides and facilitating purification of the expressed protein by means of methotrexate-bound affinity chromatography and by making use of the enzyme activity. Fifteen genes encoding different lengths of polypeptides of 5 to 44 amino acids were chemically synthesized and introduced into expression vectors, pTP70-1 or its derivatives. All the polypeptide genes were efficiently expressed in Escherichia coli cells as fusion proteins which show DHFR activity. The respective fusion proteins were highly purified from cell-free extracts by monitoring the DHFR activity at each purification step. The use of methotrexate-bound affinity chromatography was very effective. In order to cut out the polypeptides, the purified fusion proteins were treated with either BrCN or site-specific protease according to the spacer sequence. The objective polypeptide was purified by means of a reversed-phase high-pressure liquid chromatography (HPLC) system. Specific cleavage of the purified fusion protein actually yielded very few peptide fragments, so the assignment and isolation of the objective polypeptide were carried out without difficulty.     

Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins

We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.     

肾炎致病原重组受体相关蛋白的表达及纯化

用pGEX载体系统体外构建了Heymann肾炎致病原受体相关蛋白(RAP)重组表达质粒,经IPTG诱导,该质粒表达的融合蛋白在大肠杆菌中得到了高效表达,其表达量达39．4％,经GST-Sephrose 4B亲和层析,得到了高度纯化,其诱导产生的抗体经蛋白质印迹法分析证明能识别肾皮质天然抗原44ku受体相关蛋白.RAP表达及纯化的成功为研究致病原病理性表型提供了有利条件．     

A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: An application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C

A new strategy is described for the production of peptides enriched with stable isotopes. Peptides of interest are expressed in Escherichia coli (E. coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin. This method yields as much as 30–100 mg/l of isotope-enriched fusion proteins in minimal media. A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni²⁺-chelating affinity chromatography. The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase. Since this enzyme is also expressed at a high level in E. coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases. In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies. This flexibility enables us to prepare peptides that are unstable in a soluble state in E. coli cells. As an example, the expression and the uniform stable isotope enrichment with ¹⁵N and/or¹³ C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins. An amide group at the C-terminus of this peptide can also be formed by our method. The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR.     

Eukaryotic expression and secretion of EGFP-labeled annexin A5

The Ca²⁺-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 μg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.     

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

死亡素与泛素在大肠杆菌中的高效融合表达

死亡素是由21个氨基酸残基组成的广谱抗菌肽。为了高效表达可溶性的死亡素,本研究利用递归式PCR（recursive PCR, rPCR）扩增了死亡素基因thanatin,并将其和家蝇Musca domestica泛素基因ubiquitin构成嵌合基因,克隆到表达载体pET-32a,再与硫氧还蛋白融合后构建表达载体pET-TRX-UBI-THA。将酶切和测序鉴定正确的质粒转化表达宿主菌BL21,经0.6 mmol/L IPTG诱导,TRX-UBI-THA融合蛋白得到了高效可溶性表达。SDS-PAGE和Western blot检测结果表明融合蛋白的分子量为28.9 kD,与预期的结果一致,表达量占菌体总蛋白的46％。Western blot分析结果显示融合蛋白能与Ni-NTA鏊合物特异性的结合,表明在融合蛋白的N-端带有6×His标签。利用C-端带有6×His标签的泛素C-端水解酶对融合蛋白进行切割,切割产物经Ni²⁺-NTA亲和柱和HPLC纯化（纯化量为5.4 mg/L）,Tricince-SDS-PAGE电泳得到单一的泛素蛋白条带。电喷雾质谱(ESI-MS)分析表明,纯化的泛素分子量为2.57 kD,与通过氨基酸预测的分子量完全一致。利用琼脂孔穴扩散法对泛素活性进行检测,结果显示纯化的泛素对大肠杆菌K12D31和金黄色葡萄球菌Staphylococcus aureus具有较强的活性抑制。本研究表明,利用泛素融合技术可以高效表达可溶性的死亡素。     

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.     

Cleavage and purification of intein fusion proteins using the Streptococcus gordonii spex system

A gram-positive bacterial expression vector using Streptococcus gordonii has been developed for expression and secretion, or surface anchoring of heterologous proteins. This system, termed Surface Protein Expression system or SPEX, has been used to express a variety of surface anchored and secreted proteins. In this study, the Mycobacterium xenopi (Mxe) GyrA intein and chitin binding domain from Bacillus circulans chitinase Al were used in conjunction with SPEX to express a fusion protein to facilitate secretion, cleavage, and purification. Streptococcus gordonii was transformed to express a secreted fusion protein consisting of a target protein with a C-terminal intein and chitin-binding domain. Two target proteins, the C-repeat region of the Streptococcus pyogenes M6 protein (M6) and the nuclease A (NucA) enzyme of Staphylococcus aureus, were expressed and tested for intein cleavage. The secreted fusion proteins were purified from culture medium by binding to chitin beads and subjected to reaction conditions to induce intein self-cleavage to release the target protein. The M6 and NucA fusion proteins were shown to bind chitin beads and elute under cleavage reaction conditions. In addition, NucA demonstrated enzyme activity both before and after intein cleavage.     

Esterase 2 from Alicyclobacillus acidocaldarius as a reporter and affinity tag for expression and single step purification of polypeptides

A novel dual function (reporter and affinity) tag system has been developed. Expression vectors have been constructed to express polypeptides in Escherichia coli cells as C-terminal fusions with esterase 2, a 34-kDa protein from Alicyclobacillus acidocaldarius. Presence of esterase allows to monitor the expression of fusion proteins spectrophotometrically or by activity staining in the polyacrylamide gels. The fusion proteins can be purified from crude bacterial extracts under non-denaturing conditions by one step affinity chromatography on Sepharose CL-6B immobilized trifluoromethyl-alkyl-ketone. The esterase carrier can be cleaved from fusion proteins by digestion with amino acid sequence-specific proteases blood coagulation factor Xa. The system has been used successfully for the expression and purification of polypeptides from different prokaryotic and eukaryotic organisms.     

Efficient high level expression of peptides and proteins as fusion proteins with the N-terminal domain of L9: application to the villin headpiece helical subdomain

The efficient expression of small to midsize polypeptides and small marginally stable proteins can be difficult. A new protein fusion system is developed to allow the expression of peptides and small proteins. The polypeptide of interest is linked via a Factor Xa cleavage sequence to the C-terminus of the N-terminal domain of the ribosomal protein L9 (NTL9). NTL9 is a small (56 residue) basic protein. The C-terminus of the protein is part of an alpha-helix which extends away from the globular structure thus additional domains can be fused without altering the fold of NTL9. NTL9 expresses at high levels, is extremely soluble, and remains fully folded over a wide temperature and pH range. The protein has a high net positive charge, facilitating purification of fusion proteins by ion exchange chromatography. NTL9 fusions can also be easily purified by reverse phase HPLC. As a test case we demonstrate the high level expression of a small, 36 residue, three helix bundle, the villin headpiece subdomain. This protein is widely used as a model system for folding studies and the development of a simple expression system should facilitate experimental studies of the subdomain. The yield of purified fusion protein is 70 mg/L of culture and the yield of purified villin headpiece subdomain is 24 mg/L of culture. We also demonstrate the use of the fusion system to express a smaller marginally folded peptide fragment of the villin headpiece domain.