Purification Of Polypeptides Research Articles

Bacterial expression and/or solid phase peptide synthesis of 20-40 amino acid long polypeptides and miniproteins, the case study of Class B GPCR ligands.

By using two different synthetic techniques several polypeptides interacting with Class B type G-protein coupled receptors were prepared. These polypeptides of different lengths (20 ≤ amino acids ≤ 40), structural and aggregation properties, were prepared both by solid phase peptide synthesis (SPPS) and E.coli bacterial expression. Their purity, synthetic yields, by-products and (15)N/(13)Clabelling characteristics were compared as function of i) the applied method, ii) amino acid length and iii) folding propensities. Their tentative yields, costs and "environmental footprints" were analyzed and found as follows. For unlabelled and short polypeptides (n= 20 aa.) the method of choice is the less environmentally friendly however, quick and effective SPPS. If the polypeptide is (un)folded and/or has no aggregation propensity, then SPPS gives relatively good yield (e.g. 14 ± 4%) and a pure product (>97%). For aggregating polypeptides production yields drop for both methods 4 ± 2% (SPPS) and 2 ± 1% (E. coli), respectively. For longer (n≥ 30 aa.) macromolecules (e.g. miniproteins) bacterial expression efficacy gets higher. Moreover biotechnology is "greener", the resulting in raw material is purer (2.8 ± 1.5 mg). All these advantages for at a lower cost: ~4 €/aa. If isotopic labelling is needed for heteronuclear NMR measurements, bacterial expression is the sole option, due to the high cost of (15)N/(13)C labelled Fmoc(Boc)-L-aa-OH starting materials needed for SPPS. In E.coli uniformly double-labelled, pure polypeptides can be obtained for less than 5-700 €/mg, regardless of the length of the polypeptide chain. Thus, chemists are encouraged to use E.coli expression systems when adequate to make not only proteins but polypeptides and miniproteins as well.

Studies on the purification of polypeptide from sika antler plate and activities of antitumor.

BackgroundWe isolated a novel monomeric peptide from antler plate polypeptide (APP) of sika deer and found that it inhibited rat breast cancer cell proliferation and telomerase activity.MethodsThe molecular mass and purity of this polypeptide was determined by ultra performance liquid chromatography (UPLC) and Bruker micOTOF OllQ TOF mass spectrometry, respectively. The full amino-acid sequence of the monomeric peptide was analyzed by sequential Edman degradation using a protein/peptide sequencer. The APP-1 markedly inhibited rat breast cancer cell proliferation as determined with an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) assay. Then, we used flow cytometry to detect the effects of the monomeric peptide on cell cycle. Relative quantitative fluorescence PCR was used to analyze the expression level telomerase reverse transcriptase (TERT).ResultsThe molecular mass and purity of this polypeptide was 10646 Da and 91.2 %. Amino acid sequence analyses indicated that the N-terminal amino-acid sequence of this monomeric peptide was: MTKLE DYLEG IVNIF HQYSV. The results showed that monomeric peptide halted most cancer cells stagnating in the G0/G1 phase. The percentage of cells in the G0/G1 is higher than control group after the monomeric peptide treatment. Relative quantitative fluorescence PCR results showed that TERT gene expression level obviously decreased after treatment with the monomeric peptide compared with control group.ConclusionsCollectively, the results suggest that this novel and monomeric APP has antitumor activities and imply that it is likely an important component of antitumor activities in antler plate polypeptide.

NMR Assignment and Secondary Structure of Coiled Coil Domain of C-terminal Myosin Binding Subunit of Myosin Phosphatase

Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

Solid phase protein chemical synthesis.

The chemical synthesis of peptides or small proteins is often an important step in many research projects and has stimulated the development of numerous chemical methodologies. The aim of this review is to give a substantial overview of the solid phase methods developed for the production or purification of polypeptides. The solid phase peptide synthesis (SPPS) technique has facilitated considerably the access to short peptides (<50 amino acids). However, its limitations for producing large homogeneous peptides have stimulated the development of solid phase covalent or non-covalent capture purification methods. The power of the native chemical ligation (NCL) reaction for protein synthesis in aqueous solution has also been adapted to the solid phase by the combination of novel linker technologies, cysteine protection strategies and thioester or N,S-acyl shift thioester surrogate chemistries. This review details pioneering studies and the most recent publications related to the solid phase chemical synthesis of large peptides and proteins.

Using Homo-Polypeptides to Study Charge-Charge Interactions in Biomolecules

Charge-charge interactions in biomolecules are important for a range of biological processes such as enzyme-substrate and ligand-receptor recognition, auto-inhibition of signaling proteins, protein folding and balancing intrinsically disordered protein (IDP) function with aggregation. In order to more thoroughly understand the roles of charge-charge interactions in these systems, we use homo-polypeptides composed of glutamic acid and lysine residues. We use a range of calorimetric (ITC) and spectroscopic (FRET, CD, NMR) techniques to measure polypeptide binding and conformation under a range of solvent conditions such as pH, ionic strength and counter ion identity. In particular, using FRET to monitor such interactions allows us to achieve extremely high levels of sensitivity, but requires us to add an external fluorophores to the polypeptides. We describe the labeling and purification of polypeptides and determine the effect of the label on the interaction by comparing the thermodynamic properties of the labeled and unlabeled polypeptides. Results of this study show that the intermolecular interaction(s) through the charged side-chains of amino acids can significantly influence the backbone conformation of polypeptides. Our overall aim is to quantify the relative contributions of enthalpy and entropy in determining interaction mechanisms in highly charged regions of proteins.

A slow gradient approach for the purification of synthetic polypeptides by reversed phase high performance liquid chromatography

Unquestionably, the purification of polypeptides by chromatographic methods is a considerable bottleneck in their preparation. Peptides synthesised by solid phase synthesis typically contain chromatographically similar impurities that complicate purification by reversed phase high performance liquid chromatography (HPLC) techniques. We report on the application of a slow gradient HPLC protocol that allows, in a single chromatographic step, the purification of hundreds of milligrammes of material. This technique was applied to an extensive collection of synthetic polypeptides some incorporating non-proteinogenic functionality. In all cases examined, the peptides were not only obtained in high purity peptides but were also recovered in multi-milligramme amounts.

Optimizing model predictive control of the chromatographic multi-column solvent gradient purification (MCSGP) process

The multi-column solvent gradient purification (MCSGP) process is a new continuous chromatographic process specifically designed for the purification of proteins and peptides. Due to its countercurrent purification principle, the performance increase with respect to the batch process can be up to 10× in productivity and even more at higher yields. Various successful applications of the MCSGP-technology have been reported, e.g. the purification of monoclonal antibodies from high-titer supernatants with ion-exchange resins and the purification of polypeptides. However, optimal MCSGP operation is a challenge and the current practice is to operate the MCSGP units at sub-optimal conditions to guarantee robustness and improve the separation performance by tuning the operating conditions off-line. As a result, systematic optimization tools like control and automation of MCSGP is of great interest in order to exploit the full economic potential of this process. An automatic control algorithm for MCSGP units that guarantees an optimal, robust operation with product purities in specification is a challenging problem because of the uncertainty related to the adsorption behavior of the mixture to be separated and the complex dynamics involved in this process, i.e. its cyclic and hybrid nature due to the inlet/outlet port switching with strong nonlinearities and delays in the feedback information. In this work, a control algorithm based on previous work for control of continuous chromatographic process is developed for the MCSGP-technology. The flow rates as well as the modifier gradients have been chosen as manipulated variables. The suitability of the controller is proven by means of two simulation case studies, i.e. the separation of monoclonal antibody variants and of a mixture of peptides.

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.

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 μg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10 −8 M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein-esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N-terminus and simultaneously, as a cleavable tag for polypeptide purification.

Quantify this! Report on a round table discussion on quantitative mass spectrometry in proteomics.

Following the success of the first round table in 2001, the Swiss Proteomic Society has organized two additional specific events during its last two meetings: a proteomic application exercise in 2002 and a round table in 2003. Such events have as their main objective to bring together, around a challenging topic in mass spectrometry, two groups of specialists, those who develop and commercialize mass spectrometry equipment and software, and expert MS users for peptidomics and proteomics studies. The first round table (Geneva, 2001) entitled "Challenges in Mass Spectrometry" was supported by brief oral presentations that stressed critical questions in the field of MS development or applications (Stöcklin and Binz, Proteomics 2002, 2, 825-827). Topics such as (i) direct analysis of complex biological samples, (ii) status and perspectives for MS investigations of noncovalent peptide-ligant interactions; (iii) is it more appropriate to have complementary instruments rather than a universal equipment, (iv) standardization and improvement of the MS signals for protein identification, (v) what would be the new generation of equipment and finally (vi) how to keep hardware and software adapted to MS up-to-date and accessible to all. For the SPS'02 meeting (Lausanne, 2002), a full session alternative event "Proteomic Application Exercise" was proposed. Two different samples were prepared and sent to the different participants: 100 micro g of snake venom (a complex mixture of peptides and proteins) and 10-20 micro g of almost pure recombinant polypeptide derived from the shrimp Penaeus vannamei carrying an heterogeneous post-translational modification (PTM). Among the 15 participants that received the samples blind, eight returned results and most of them were asked to present their results emphasizing the strategy, the manpower and the instrumentation used during the congress (Binz et. al., Proteomics 2003, 3, 1562-1566). It appeared that for the snake venom extract, the quality of the results was not particularly dependant on the strategy used, as all approaches allowed Lication of identification of a certain number of protein families. The genus of the snake was identified in most cases, but the species was ambiguous. Surprisingly, the precise identification of the recombinant almost pure polypeptides appeared to be much more complicated than expected as only one group reported the full sequence. Finally the SPS'03 meeting reported here included a round table on the difficult and challenging task of "Quantification by Mass Spectrometry", a discussion sustained by four selected oral presentations on the use of stable isotopes, electrospray ionization versus matrix-assisted laser desorption/ionization approaches to quantify peptides and proteins in biological fluids, the handling of differential two-dimensional liquid chromatography tandem mass spectrometry data resulting from high throughput experiments, and the quantitative analysis of PTMs. During these three events at the SPS meetings, the impressive quality and quantity of exchanges between the developers and providers of mass spectrometry equipment and software, expert users and the audience, were a key element for the success of these fruitful events and will have definitively paved the way for future round tables and challenging exercises at SPS meetings.

Inexpensive one-step purification of polypeptides expressed in Escherichia coli as fusions with the family 9 carbohydrate-binding module of xylanase 10A from T. maritima

A novel inexpensive affinity purification technology is described based on recombinant expression in Escherichia coli of the polypeptide or protein target fused through its N-terminus to TmXyn10ACBM9-2 (CBM9), the C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima. Measured association constants ( K a) for adsorption of CBM9 to insoluble allomorphs of cellulose are between 2×10 5 and 8×10 6 M −1. CBM9 also binds a range of soluble sugars, including glucose. As a result, a 1 M glucose solution is effective in eluting CBM9 and CBM9-tagged fusion proteins from a very inexpensive commercially-available cellulose-based capture column. A processing site is encoded at the C-terminus of the tag to facilitate its rapid and quantitative removal by Factor X a to recover the desired target protein sequence following affinity purification. Fusion of the CBM9 affinity tag to the N-terminus of green fluorescent protein (GFP) from the jellyfish, Aquorin victoria, is shown to yield >200 mg l −1 of expressed soluble fusion protein that can be affinity separated from clarified cell lysate to a purity of >95% at a yield of 86%.

Statistical analysis on protein-protein interface in crystals: Specific and non-specific interfaces are differentially distributed

The distribution of contact areas, or fractions of contacting, of protein-protein interfaces in crystals of pure polypeptides contains two components: a major exponential distribution and a minor flatter distribution. Suppose the two components belong to specific and non-specific contacts, respectively, then the probability of a contact with a given area, or fraction of contacting, can be estimated. By dividing the whole database into two sub-databases, one of them is known to contain more specific contacts than the other, this hypothesis is confirmed and it is also proved that the fraction of contacting is more effective than the contact area on discriminating specific and non-specific contacts in protein crystals.

Purification of Glutamate Dehydrogenase Isoenzymes and Characterization of Their Substrate Specificities

Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, α, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06–0.8 mM of each of UTP, ATP, GTP, and CTP, 0–100.0 mM NH4Cl, 0–50.0 mM α-ketoglutaratr (α-KG), 0–0.2 mM NADH, 0–10.0 mM CaCl2 5 units of DNase 1, antibiotics, and ∼5 µg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16°C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and α-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for the isoenzymes in RNA synthesis reaction. The product RNA was up to 2 µg µg−1 GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [α-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

O-Glycosylation of a Recombinant Carbohydrate-Binding Module Mutant Secreted by Pichia pastoris

Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly–, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man₁ to Man₄. MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly– and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain α-1,2-, α-1,3-, and α-1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly– a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.

Covalent capture purification of polypeptides after SPPS via a linker removable under very mild conditions

The covalent purification of polypeptides possessing an N-terminal cysteine or threonine residue via formation of a thiazolidine or oxazolidine with an aldehyde-functionalized-resin has been successfully demonstrated. To extend the applicability of this approach to any possible N-terminal residue, a special linker derived from ( S)-4-amino-2-hydroxy-butyric acid was incorporated into peptidyl-resin. This linker represents the connecting point between the capture unit (cysteine) useful for the isolation of the desired polypeptide and the desired N-terminus. The target polypeptide was recovered by periodate oxidation, which cleaved the covalent bond between the linker and the last residue of polypeptide under very mild conditions.

Isolation and purification of BV?-2H from bee venom and analysis of its biological action

The medical use of bee venom for rheumatoid arthritis (RA) has a very long tradition. In this study, isolation and purification of polypeptides from bee venom were carried out on sephadex chromatography, heparin sepharose CL-6B chromatography and HPLC. Several fractions were extracted, and their effects on activation of splenocyte and THP-1 cell were studied. The inhibitory fraction was selected for further studies. Finally, BV I -2H that the HPLC elution profiles was a single peak was isolated by C8 column. ESIMS detection results showed that BV I -2H was a fraction of bee venom, and the molecular weight of the major component was 644.8. BV I -2H could inhibit ConA-induced splenocyte proliferation, IL-1 production and interfere with splenocyte cycle in mice. Moreover, BV I -2H could inhibit PMA-induced TNFα production in THP-1 cells, which was due to its inhibitory effects on TNFα mRNA expression and protein phosphorylation of IκBα. Our studies indicated that BV I -2H was one of the anti-inflammatory components of bee venom.

Covalent capture: a new tool for the purification of synthetic and recombinant polypeptides

Background: Purification of polypeptides and proteins derived from recombinant DNA techniques and of long synthetic polypeptides often represents a challenge. Affinity methods exist, but generally require addition of a large recognition unit to the target protein and use of expensive purification media. Use of large units is dictated by the characteristics of non-covalent complexes, where the energy necessary to form the complex derives from the sum of multiple weak energy interactions. Covalent interactions in contrast are of high energy, even when only a few bonds are formed. We decided to explore the use of the reversible covalent bond formed between N-terminal cysteine and threonine residues with an aldehyde as a method of protein purification. Results: A series of test peptides with N-terminal cysteine and threonine were captured by a polyethyleneglycol–polyacrylamide resin to which an aldehyde function had been grafted. Peptides with other amino acids at the N-terminus did not interact with the resin. A recombinant polypeptide with N-terminal cysteine was purified to 90% purity in one step. Polypeptides were eluted from the resin simply by adding a hydroxylamine derivative, which reacts with aldehyde functions to form an oxime. Conclusions: Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this ‘covalent capture’ approach.

Design of high-throughput methods of protein production for structural biology

I greatly appreciate interacting with Peter Schultz, Scott Lesley and Marc Nasoff at the Genomics Institute for the Novartis Research Foundation and Mark Knuth and Ron Swanson at Syrrx, Inc., where much of this work is currently being pursued. I also thank Jim Graziano, Chris Lee, Mike Hanson and Marianne Patch for helpful suggestions on this manuscript.

Mapping epitopes to distinct regions of the extracellular domain of endoglin using bacterially expressed recombinant fragments.

Endoglin (CD105) is a homodimeric cell surface component of the TGF-beta 1 receptor complex, which is expressed at high levels on vascular endothelium and at lower levels on activated monocytes. It is also the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1. To date, each family has a distinct endoglin mutation, most of which generate premature stop codons. The purpose of the current study was to identify monoclonal antibodies capable of binding to normal and mutated forms of the protein. We generated stable transfectants of full-length human endoglin in murine fibroblasts and engineered and expressed in bacteria several fragments of the extracellular domain. Relatively pure polypeptides were recovered with good yield from inclusion bodies and were tested by ELISA and Western blot; 11 monoclonal antibodies were shown to react specifically with the endoglin transfectants. Ten of these monoclonal antibodies reacted with the bacterial fragments, and their epitopes were assigned to 3 distinct regions of endoglin. Monoclonal antibodies P3D1, TEC4 and GRE reacted with the N-terminal region of 204 amino acids encoded by exons 1 to 5. Monoclonal antibodies P4A4, 44G4, E-9, MAEND3 and PN-E2 all bound to a region of 54 amino acids encoded mostly by exon 7. Monoclonal antibodies CLE4 and RMAC8 reacted with the C-terminal region of the extracellular domain, coded for by exons 8 to 12. Knowing the localization of these epitopes will facilitate the structural and functional analysis of normal and mutated forms of endoglin.

Characterization of the main egg envelope proteins of the sea bass Dicentrarchus labrax L. (teleostea, serranidae)

Fish eggs are surrounded by a resistant acellular coat commonly called the chorion or zona radiata. This study characterizes the eggshell proteinaceous content of unfertilized eggs of the sea bass Dicentrarchus labrax, with a view to the preparation of immunogens. Solubilization of the purified eggshells was achieved in 8 M urea followed by one- and two-dimensional gel electrophoresis. Glycoproteins were detected using concanavalin-A in one and two-dimensional gels, and the principal glycoproteins had a molecular weight of 47 kDa and 170 kDa. Partial purification of a few polypeptides in the 45 kDa to 55 kDa range was achieved by gel filtration chromatography. Although whole eggshells were relatively insoluble even in 8 M urea, partial purification of these polypeptides enable them to dissolve completely in solutions at low ionic strength.