Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying.

Late embryogenesis abundant (LEA) proteins have been repeatedly implicated in the acquisition of desiccation tolerance in angiosperm seed embryos. However, the mechanism(s) by which protection occurs is not well understood. While the Group 1 LEA proteins are predicted to be largely unordered in solution, there is strong evidence that upon drying these proteins undergo a structural transition that leads to an increase in alpha-helical content. Several studies also suggest there is a direct interaction between Group 1 LEA proteins and other molecules in the cytoplasm that may be critical for the establishment of desiccation tolerance during embryo maturation. We have produced a recombinant Group 1 LEA protein and show that it is capable of protecting the enzyme lactate dehydrogenase from the deleterious effects of drying. We have also evaluated the ability of various altered recombinant Group 1 LEA proteins to protect in the same assay. Our results suggest that the highly conserved 20 amino acid Group 1 LEA signature motif is not required for protection in our in vitro assay. However, introduction of two juxtaposed proline residues into an N-terminal helical domain predicted to exist in the hydrated structure significantly compromises the ability of the recombinant protein to provide protection from drying. These results suggest that the N-terminal domain of Group 1 LEA proteins may be important for proper folding during dehydration.     

A simplified method for purification of recombinant soluble DnaA proteins

An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.     

A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 m urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR(R) predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.     

Two different late embryogenesis abundant proteins from Arabidopsis thaliana contain specific domains that inhibit Escherichia coli growth

Late embryogenesis abundant (LEA) proteins constitute a set of proteins widespread in the plant kingdom that show common physicochemical properties such as high hydrophilicity and high content of small amino acid residues such as glycine, alanine, and serine. Typically, these proteins accumulate in response to water deficit conditions imposed by the environment or during plant normal development. In this work, we show that the over-expression in Escherichia coli of proteins of the LEA 2 and the LEA 4 families from Arabidopsis thaliana leads to inhibition of bacterial growth and that this effect is dependent on discrete regions of the proteins. Our data indicate that their antimicrobial effect is achieved through their interaction with intracellular targets. The relevance of the cationic nature and the predicted structural organization of particular protein domains in this detrimental effect on the bacteria growth process is discussed.     

A novel method for the purification of low soluble recombinant C-type lectin proteins

Snake venoms contain a complex mixture of many biological molecules including proteins. The purification of recombinant proteins is a key step in studying their function and structure with affinity chromatography as the common method used in their purification. In bacterial expression systems, hydrophobic recombinant proteins are usually precipitated into inclusion bodies, and contaminants are typically associated with tagged proteins after purification. The purpose of this study was to develop a procedure to purify hydrophobic recombinant proteins without an affinity tag. Snake venom mature C-type lectin-like proteins (CLPs) with a tag were cloned, expressed, and purified by repeated sonication and wash steps. The effects of the signal peptide on the expression and solubility of the recombinant protein were investigated. The CLPs in washed inclusion bodies were solubilized and refolded by dialysis. The CLPs without a tag were successfully purified with a yield 38 times higher than the traditional method, and inhibited blood platelet aggregation with an IC(50) of 100.57μM in whole blood. This novel procedure is a rapid, and inexpensive method to purify functional recombinant hydrophobic CLPs from snake venoms useful in the development of drug therapies.     

Boost Protein Expression through Co-Expression of LEA-Like Peptide in Escherichia coli

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.     

Structural disorder throws new light on moonlighting

A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins (IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.     

Biochemical and structural characterization of an endoplasmic reticulum-localized late embryogenesis abundant (LEA) protein from the liverwort Marchantia polymorpha

Late embryogenesis abundant (LEA) proteins, which accumulate to high levels in seeds during late maturation, are associated with desiccation tolerance. A member of the LEA protein family was found in cultured cells of the liverwort Marchantia polymorpha; preculture treatment of these cells with 0.5 M sucrose medium led to their acquisition of desiccation tolerance. We characterized this preculture-induced LEA protein, designated as MpLEA1. MpLEA1 is predominantly hydrophilic with a few hydrophobic residues that may represent its putative signal peptide. The protein also contains a putative endoplasmic reticulum (ER) retention sequence, HEEL, at the C-terminus. Microscopic observations indicated that GFP-fused MpLEA1 was mainly localized in the ER. The recombinant protein MpLEA1 is intrinsically disordered in solution. On drying, MpLEA1 shifted predominantly toward α-helices from random coils. Such changes in conformation are a typical feature of the group 3 LEA proteins. Recombinant MpLEA1 prevented the aggregation of α-casein during desiccation–rehydration events, suggesting that MpLEA1 exerts anti-aggregation activity against desiccation-sensitive proteins by functioning as a “molecular shield”. Moreover, the anti-aggregation activity of MpLEA1 was ten times greater than that of BSA or insect LEA proteins, which are known to prevent aggregation on drying. Here, we show that an ER-localized LEA protein, MpLEA1, possesses biochemical and structural features specific to group 3 LEA proteins.     

A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins

We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes.     

NMR relaxation studies on the hydrate layer of intrinsically unstructured proteins

Intrinsically unstructured/disordered proteins (IUPs) exist in a disordered and largely solvent-exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention. Here we demonstrate that the hydration of IUPs can be adequately approached by a technique so far unexplored with respect to IUPs, solid-state NMR relaxation measurements. This technique provides quantitative information on various features of hydrate water bound to these proteins. By freezing nonhydrate (bulk) water out, we have been able to measure free induction decays pertaining to protons of bound water from which the amount of hydrate water, its activation energy, and correlation times could be calculated. Thus, for three IUPs, the first inhibitory domain of calpastatin, microtubule-associated protein 2c, and plant dehydrin early responsive to dehydration 10, we demonstrate that they bind a significantly larger amount of water than globular proteins, whereas their suboptimal hydration and relaxation parameters are correlated with their differing modes of function. The theoretical treatment and experimental approach presented in this article may have general utility in characterizing proteins that belong to this novel structural class.     

Both plant and animal LEA proteins act as kinetic stabilisers of polyglutamine-dependent protein aggregation

LEA (late embryogenesis abundant) proteins are intrinsically disordered proteins that contribute to stress tolerance in plants and invertebrates. Here we show that, when both plant and animal LEA proteins are co-expressed in mammalian cells with self-aggregating polyglutamine (polyQ) proteins, they reduce aggregation in a time-dependent fashion, showing more protection at early time points. A similar effect was also observed in vitro, where recombinant LEA proteins were able to slow the rate of polyQ aggregation, but not abolish it altogether. Thus, LEA proteins act as kinetic stabilisers of aggregating proteins, a novel function in protein homeostasis consistent with a proposed role as molecular shields.     

Purification of dual-tagged intact recombinant proteins

Large-scale purification of recombinant proteins has been used extensively to assist numerous protein studies, including investigation of function, substrate identification and protein-protein interaction of low abundance proteins. Genetic fusion of affinity tags to these proteins has also been widely used for ease of purification by affinity chromatography. However, this technique sometimes yields unstable and degraded protein products limiting its application. In this study, we show a facile and straightforward method of dual-tagged recombinant protein purification that eliminates contamination by degraded protein products. A 6His-containing BamHI-HindIII fragment from pQE12 was ligated into the pGEX-KG BamHI-HindIII fragment and the protein of interest (p25(nck5a), which is highly susceptible to proteolytic degradation when expressed and purified from bacteria) was cloned into the BamHI site without a termination codon. The resulting plasmid construct, designated as pGST-p25(nck5a)-6His, with GST at the N-terminal and 6His at the C-terminal was expressed in Escherichia coli DH5alpha and purified using a two-step procedure. We show that using Ni(2+)-NTA chromatography as a first purification step and GSH-agarose chromatography as a second step, rather than vice-versa, yields a highly purified intact protein that is free of any contaminating degraded protein product. The purified fusion protein is soluble and fully active.     

A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to high concentrations during the late stages of seeds development, which are integral to desiccation tolerance. LEA proteins also play a protective role under other abiotic stresses. We analyzed in silico a maize protein predicted to be highly hydrophilic and intrinsically disordered. This prediction was experimentally corroborated by solubility assays under denaturing conditions. Based on its amino acid sequence, we propose that this protein belongs to group four of the LEA proteins. The accumulation pattern of this protein was similar to that of dehydrins during the desiccation process that takes place during seed development. This protein was induced by exogenous abscisic acid in immature embryos, but during imbibition was down-regulated by gibberellins. It was also induced in maize roots under osmotic stress. So far, this is the first member of the LEA proteins belonging to group four to be characterized in maize, and it plays a role in the response to osmotic stress.     

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa.     

Production of recombinant proteins in plant root exudates.

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.     

Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.     

Simultaneous phase transition of ELP tagged molecules and free ELP: an efficient and reversible capture system

In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of molecules present at very low concentrations in complex mixtures. The method makes use of an elastin-like polypeptide (ELP) as a coaggregant for the capture of an ELP tagged recombinant protein present at concentrations as low as 10 pM, with a recovery higher than 90%. This coaggregation process was found to be independent of the concentration, at least up to 10 pM concentration of the ELP tagged protein. The coaggregation process is highly specific as was demonstrated by spiking crude cell lysate with the ELP tagged recombinant protein to a final concentration of 1 nM and recovering more than 80% of it to a high level of purity. The method should be particularly useful for high-throughput proteomic studies, where small amounts of poorly expressed proteins could be recovered for analysis by mass spectrometry. In a more general context, the concept presented in this paper provides a method that is highly efficient, specific, and fully reversible, which should render it useful in areas other than recombinant protein purification.