Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

Automated purification of recombinant proteins: combining high-throughput with high yield

Protein crystallography, mapping protein interactions, and other functional genomic approaches require purifying many different proteins, each of sufficient yield and homogeneity, for subsequent high-throughput applications. To fill this requirement efficiently, there is a need to develop robust, automated, high-throughput protein expression, and purification processes. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli (E. coli). The first is a filtration separation protocol in which proteins of interest are expressed in a large volume, 800 ml of E. coli cultures, then isolated by filtration purification using Ni-NTA-Agarose (Qiagen). The second is a smaller scale magnetic separation method in which proteins of interest are expressed in a small volume, 25 ml, of E. coli cultures then isolated using a 96-well purification system with MagneHis Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins, about 8 microg of purified protein per optical density unit of bacterial culture measured at 600 nm. We discuss advantages and limitations of these automated workflows, which can provide proteins with more than 90% purity and yields in the range of 100 microg to 45 mg per purification run, as well as strategies for optimizing these protocols.     

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5'-end and XhoI at the 3'-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His(6)-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His(6))-tagged Smt3. Smt3 is the yeast SUMO protein. His(6)-Smt3-X was purified by Ni(2+) resin. Removal of His(6)-Smt3 was performed on the Ni(2+) resin by an engineered SUMO protease, His(6)-Ulp1(403-621)-His(6). Because of its dual His(6) tags, His(6)-Ulp1(403-621)-His(6) exhibits a high affinity for Ni(2) resin and associates with Ni(2+) resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni(2+)-resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.     

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

Production and purification of an analog of glucagon-like peptide-1 by auto-induction and on-column cleavage in <Emphasis Type="Italic">Escherichia coli</Emphasis>

As a promising type 2 anti-diabetic agent, glucagon-like peptide-1 (GLP-1) is attracting more and more interest. Mutated GLP-1 (mGLP-1) is an analog of native GLP-1. To facilitate the production and purification of mGLP-1, auto-induction and on-column cleavage was employed in this study. By using auto-induction system, after 24 h of shaking culture, about 12.6 g wet bacterial cells could be obtained from 1 l medium, and this was about 3.6 times more than that of the IPTG-induction group. After disruption and centrifugation, the fusion protein was directly purified and cleaved on Ni–Sepharose 6 Fast Flow column. Then, RESOURCE15 RPC column was used for further purification. By using these two steps of purification, about 1.58 mg of mGLP-1 with the purity of up to 98% could be obtained from 1 g wet bacterial cells. In the bioactivity study, mGLP-1 displayed a significant and dose-dependent glucose-lowering activity. These results suggested that auto-induction and on-column cleavage could facilitate the production and purification of mGLP-1. These methods could also be applied to the preparation of other proteins and peptides.     

Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination

A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels [Sanville Millard, C. et al. 2003. Protein Expr. Purif. 29, 311-320]. The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies. We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction. Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteins in good yield. Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis. Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two. In the preceding paper [Zhao et al., this issue, pp. 87-93], the approach is further extended to production of [U-15N]- or [U-13C, U-15N]-labeled proteins. The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.     

Expression and simple,one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in <Emphasis Type="Italic">Escherichia coli</Emphasis> and their interaction with protoporphyrin IX

The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap₃A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His₆-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (K _m value for Ap₃A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.     

Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production

As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.     

Refolding and purification of recombinant human PDE7A expressed in Escherichia coli as inclusion bodies

We have investigated the refolding and purification of the catalytic domain of human 3',5'-cyclic nucleotide phosphodiesterase 7A1 (PDE7A1) expressed in Escherichia coli. A cDNA encoding an N-terminal-truncated PDE7A1(147-482-His) was amplified by RT-PCR from human peripheral blood cells and inserted into the vector pET21-C for bacterial expression of the enzyme fused to a C-terminal His-tag. The PDE was found to be expressed in the form of inclusion bodies which could be refolded to an active enzyme in buffer containing high concentrations of arginine hydrochloride, ethylene glycol, and magnesium chloride at pH 8.5. The PDE7A1(147-482-His) construct could be purified after dialysis and concentration steps by either Zn2+-IDA-Sepharose chromatography or ResourceQ ion-exchange chromatography to homogeneity. In comparison to the metal-chelate column, the ResourceQ purification resulted in a distinctly better yield and enrichment of the protein. Both the Vmax (0.46 micromol. min(-1). mg(-1) ) and the K(m) (0.1 microM) of the purified enzyme were found to be comparable with published data for native or recombinant catalytically active expressed PDE7A1. Using SDS/PAGE, a molecular mass of 39 kDa was determined (theoretical value 38.783 kDa). As known from several other mammalian PDEs, size-exclusion chromatography using refolded PDE7A1(147-482-His) indicated the formation of dimers. The purified enzyme was soluble at concentrations up to 100 microg/ml. A further increase of protein concentration resulted, however, in precipitation of the enzyme.     

A combined approach to improving large-scale production of tobacco etch virus protease

Tobacco etch virus NIa proteinase (TEV protease) is an important tool for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of approximately 400 mg/L of expression culture (approximately 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (approximately 12 mg pure protease fusion per gram of E. coli cell paste) are also reported.     

A rapid and universal tandem-purification strategy for recombinant proteins

A major goal in the production of therapeutic proteins, subunit vaccines, as well as recombinant proteins needed for structure determination and structural proteomics is their recovery in a pure and functional state using the simplest purification procedures. Here, we report the design and use of a novel tandem (His)(6)-calmodulin (HiCaM) fusion tag that combines two distinct purification strategies, namely, immobilized metal affinity (IMAC) and hydrophobic interaction chromatography (HIC), in a simple two-step procedure. Two model constructs were generated by fusing the HiCaM purification tag to the N terminus of either the enhanced green fluorescent protein (eGFP) or the human tumor suppressor protein p53. These fusion constructs were abundantly expressed in Escherichia coli and rapidly purified from cleared lysates by tandem IMAC/HIC to near homogeneity under native conditions. Cleavage at a thrombin recognition site between the HiCaM-tag and the constructs readily produced untagged, functional versions of eGFP and human p53 that were >97% pure. The HiCaM purification strategy is rapid, makes use of widely available, high-capacity, and inexpensive matrices, and therefore represents an excellent approach for large-scale purification of recombinant proteins as well as small-scale protein array designs.     

Rapid,robotic, small‐scale protein production for NMR screening and structure determination

Three‐dimensional protein structure determination is a costly process due in part to the low success rate within groups of potential targets. Conventional validation methods eliminate the vast majority of proteins from further consideration through a time‐consuming succession of screens for expression, solubility, purification, and folding. False negatives at each stage incur unwarranted reductions in the overall success rate. We developed a semi‐automated protocol for isotopically‐labeled protein production using the Maxwell‐16, a commercially available bench top robot, that allows for single‐step target screening by 2D NMR. In the span of a week, one person can express, purify, and screen 48 different ¹⁵N‐labeled proteins, accelerating the validation process by more than 10‐fold. The yield from a single channel of the Maxwell‐16 is sufficient for acquisition of a high‐quality 2D ¹H‐¹⁵N‐HSQC spectrum using a 3‐mm sample cell and 5‐mm cryogenic NMR probe. Maxwell‐16 screening of a control group of proteins reproduced previous validation results from conventional small‐scale expression screening and large‐scale production approaches currently employed by our structural genomics pipeline. Analysis of 18 new protein constructs identified two potential structure targets that included the second PDZ domain of human Par‐3. To further demonstrate the broad utility of this production strategy, we solved the PDZ2 NMR structure using [U‐¹⁵N,¹³C] protein prepared using the Maxwell‐16. This novel semi‐automated protein production protocol reduces the time and cost associated with NMR structure determination by eliminating unnecessary screening and scale‐up steps.     

Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis

In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.     

Production and characterization of biologically active human GM-CSF secreted by genetically modified plant cells

Human granulocyte-macrophage colony-stimulating factor (GM-CSF), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. The GM-CSF cDNA was carried by a binary vector under the control of the CaMV 35S promoter and the T7 terminator. In addition, a 5'-nontranslated region from the tobacco etch virus (TEV leader sequence) was fused to the N-terminal end of the GM-CSF transgene. For ease of purification, a 6-His tag was added to the 3' end of the GM-CSF cDNA. Addition of the TEV leader sequence increased protein production more than twofold compared to non-TEV controls. Initial batch cultivation studies indicated a maximum of 250 microg/L extracellular and 150 microg/L intracellular GM-CSF. Western blot analysis detected multiple peptides with masses from 14 to 30 kDa in the extracellular medium. The plant-produced GM-CSF was biologically active and could be bound to a nickel affinity matrix, indicating that both the receptor-binding region and the 6-His tag were functional. The batch production of GM-CSF was compared with the production of other recombinant proteins secreted by transformed tobacco cells. The recovery of secreted GM-CSF was increased by the addition of stabilizing proteins and by increasing salt in the growth medium to physiological levels.     

Reliable scale-up of membrane protein over-expression by bacterial auto-induction: From microwell plates to pilot scale fermentations

The production of well-ordered crystals of membrane proteins for structural investigation by X-ray diffraction typically requires extensive crystallization trials and may involve the screening of multiple detergents, lipids and other additives. Purification of sufficient amounts of protein for such trials is hampered by the fact that even when over-expressed, membrane proteins represent only a small percentage of the total protein content of bacteria. Fermentation-scale cultures of cells are therefore usually required. To maximize the efficiency and reduce the cost of such cultures, in the UK Membrane Protein Structure Initiative we have systematically investigated the use of auto-induction as an alternative to induction of expression with isopropyl-β-D-thiogalactoside. We report here the benefits of first optimizing expression on a multiwell plate scale by systematically varying the concentrations of glucose, glycerol, lactose and succinate present in the auto-induction medium. For subsequent scale-up, comparison of isopropyl-β-D-thiogalactoside induction in shake-flasks with auto-induction in shake-flasks and in 1L fermenters without and with control of pH and aeration revealed that highest yields of target protein were obtained using the latter culture conditions. However, analysis of the time-course of expression highlighted the importance of choosing the correct time for harvest. The high yields of target protein that can be obtained in a single batch by auto-induction, performed on a 30 l scale in a fermenter, obviate batch-to-batch variations that can add an unwanted variable to crystallization screening experiments. The approach described should therefore be of great utility for membrane protein production for structural studies.     

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.     

High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography

A high-throughput protein purification strategy using the polycationic Z(basic) tag has been developed. In order for the strategy to be useful both for soluble and less soluble proteins, a denaturating agent, urea, was used in all purification steps. First, four target proteins were genetically fused to the purification tag, Z(basic). These protein constructs were purified by cation exchange chromatography and eluted using a salt gradient. From the data achieved, a purification strategy was planned including stepwise elution to enable parallel protein purification using a laboratory robot. A protocol that includes all steps, equilibration of the chromatography resin, load of sample, wash, and elution, all without any manual handling steps, was handled by the laboratory robot. The program allows automated purification giving milligram amounts of pure recombinant protein of up to 60 cell lysates. In this study 22 different protein constructs, with different characteristics regarding pI and solubility, were successfully purified by the laboratory robot. The data show that Z(basic) can be used as a general purification tag also under denaturating conditions. Moreover, the strategy enables purification of proteins with different pI and solubility using ion exchange chromatography (IEXC). The procedure is highly reproducible and allows for high protein yield and purity and is therefore a good complement to the commonly used His(6)-tag.     

High-level expression and single-step purification of human tryptophanyl-tRNA synthetase.

Human trpS gene was cloned into the expression vector pET-24a(+) to yield pET-24a(+)-HTrpRS, which could direct the synthesis of a mammalian derived protein in Escherichia coli BL21-CodonPlus(DE3)-RIL. The vector allows overproduction and single-step purification of His(6)-tagged human tryptophanyl-tRNA synthetase by the facilitation of metal (Ni(2+)) chelate affinity chromatography. The expression level of human TrpRS was about 40% of total cell proteins after isopropyl beta-D-thiogalactoside induction. The overproduced human TrpRS-His(6) could be purified to homogeneity within 2 h and about 24 mg purified enzyme could be obtained from 400 ml cell culture. The His(6) tag at C terminus had little effect on the binding ability of its substrates.     

Production in two-liter beverage bottles of proteins for NMR structure determination labeled with either 15N- or 13C-15N

The use of 2-L polyethylene terephthalate beverage bottles as a bacterial culture vessel has been recently introduced as an enabling technology for high-throughput structural biology [Sanville Millard, C. et al., 2003. Protein Express. Purif. 29, 311-320]. In the article following this one [Stols et al., this issue, pp. 95-102], this approach was elaborated for selenomethionine labeling used for multiwavelength anomalous dispersion phasing in the X-ray crystallographic determinations of protein structure. Herein, we report an effective and reproducible schedule for uniform 15N- and 13C-labeling of recombinant proteins in 2-L beverage bottles for structural determination by NMR spectroscopy. As an example, three target proteins selected from Arabidopsis thaliana were expressed in Escherichia coli Rosetta (DE3)/pLysS from a T7-based expression vector, purified, and characterized by electrospray ionization mass spectrometry and NMR analysis by 1H-15N heteronuclear single quantum correlation spectroscopy. The results show that expressions in the unlabeled medium provide a suitable control for estimation of the level of production of the labeled protein. Mass spectral characterizations show that the purified proteins contained a level of isotopic incorporation equivalent to the isotopically labeled materials initially present in the growth medium, while NMR analysis of the [U-15N]-labeled proteins provided a convenient method to assess the solution state properties of the target protein prior to production of a more costly double-labeled sample.