Protein Purification Protocols Research Articles

Design of Protein Purification Pathways: Application to the Proteome ofHaemophilus influenzaeUsing Heparin Chromatography

The design of efficient protein purification protocols is a scientific challenge and can be facilitated by the use of master protein enrichment or purification steps. A master purification step is in principle a list including the major proteins expected to be present in the various fractions collected from a particular enrichment process. Consideration of a master step in the design of a purification pathway can reduce the time and effort usually invested in “trial and error” approaches. Moreover, master purification steps will certainly become valuable tools in the isolation of proteins today designated as hypothetical or predicted coding regions, resulting from the sequencing of the various genomes, and for which no information on their purification exists. The construction of such a master step consists of performance of the protein separation by the particular chromatographic method, analysis by two-dimensional polyacrylamide gel electrophoresis, and identification by mass spectrometry of the proteins present in each fraction. This can be efficiently accomplished now thanks to developments in two-dimensional gel technology and large-scale sample throughput mass spectrometry. Here we present an example of construction of a master protein enrichment step using the soluble protein fraction of the bacteriumHaemophilus influenzaeand applying Heparin chromatography.

24] Identification of phosphorylation sites in human MDR1 P-glycoprotein

This chapter discusses the methodology utilized in identification of phosphorylation sites in human multidrug resistance (MDR)-1 P-glycoprotein (Pgp). The procedure represents an amalgam of the protocols for protein and peptide purification, adapted to suit the particular characteristics of Pgp. Overexpression of Pgp is the best characterized experimental mechanism of multidrug resistance (MDR) and has been implicated in several studies as an important factor in clinical resistance to chemotherapeutic drugs. A large body of evidence based on pharmacologic, biochemical, and molecular approaches has suggested that phosphorylation of Pgp plays a modulatory role in drug transport. In vivo sites require 32 p-labeling of cells on an impractically large scale. Membrane associated Pgp is utilized as substrate rather than purified Pgp to retain Pgp in a more native conformation and not expose irrelevant sites to the exogenous kinase and to avoid solubility problems

Protein purification in multicompartment electrolyzers with isoelectric membranes

Preparative purification of proteins under isoelectric conditions is reviewed, with particular regard to novel equipment, a multicompartment electrolyzer with isoelectric membranes, which can capture any desired protein into an isoelectric trap as the sole, ultra-pure component. This novel machine is based on the Immobiline chemistry, i.e. the novel generation of non-amphoteric buffers, based on the chemistry of acrylamides, which can be insolubilized onto polyacrylamide supports. After a description of the instrument and of its performance, a number of protein purification protocols are described, leading to truly homogeneous (by the most stringent criterion of surface charge) protein fractions. Such a high charge purity has been found to be often a fundamental prerequisite for the growth of protein crystals. Interfacing the electrolyzer with mass spectrometry has permitted the decoding of the structure of minor components generated from a parental molecule, especially ones having a higher p I. It was found that these species were often generated either by proteolytic cleavage or by the formation of a trisulphide bridge between two Cys residues. A unique application of the electrolyzer is finally described: its use as an immobilized enzyme reactor under an electric field. The performance of this reaction is outstanding, in that the kinetic parameters of the immobilized enzyme are identical to those of a free enzyme form.

Micropreparative High Resolution Purification of Proteins by a Combination of Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis, Isoelectric Focusing, and Membrane Blotting

We report a simple, economical, and efficient protocol for protein purification from cells. First, proteins of cell lysates were separated by standard sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted to protein-blotting membrane. The blots were stained with Coomassie blue or developed by immunoblotting to visualize specific proteins. The bands corresponding to those visible by immunoblotting were excised from the dye-stained blots and subjected to isoelectric focusing. The focused gel was stained with Coomassie blue. Finally, the stained bands were excised and subjected to another SDS–PAGE separation and electrotransferred back to protein-blotting membrane. At this stage, the purified proteins were suitable for microsequencing. We have tested the feasibility of this novel technique by purifying proteins with molecular weights ranging from 19 to 100 kDa from a lysate ofSarcocystis neurona,the etiologic agent of equine protozoal myeloencephalitis. The purity of proteins was demonstrated by reverse-phase high-performance liquid chromatography. Partial sequences of these purified proteins were obtained by N-terminal or digestive sequencing.

Book Review: Methods in Molecular Biology. Vol. 59, Protein Purification Protocols. Edited by S. Doonan

Book Review: Methods in Molecular Biology. Vol. 59, Protein Purification Protocols. Edited by S. Doonan

Dye-affinity techniques for bioprocessing: recent developments.

Textile or triazine dyes play an important role as affinity ligands in protein purification. Each step of the protein purification protocol can be divided into three stages, partitioning between two phases, separation of these phases and recovery of the target protein from the enriched phase. Now developments in dye-affinity techniques are discussed emphasizing the innovations in all three stages of the protein purification process. Dye-affinity chromatography has become a routine step in protein purification. New dyes have been developed and used successfully in both traditional chromatographic mode and new modes like affinity precipitation, polymer aqueous two-phase partitioning or expanded bed chromatography. The specificity of dye techniques has been increased by both purposeful designing of new dyes and decreasing non-specific protein-dye interactions with polymer shielding. One can envisage further development and ramification of dye-affinity techniques in protein purification.

Purification of a ligand for the EPH-like receptor HEK using a biosensor-based affinity detection approach.

Advances in screening technologies allowing the identification of growth factor receptors solely by virtue of DNA or protein sequence comparison call for novel methods to isolate corresponding ligand growth factors. The EPH-like receptor tyrosine kinase (RTK) HEK (human EPH-like kinase) was identified previously as a membrane antigen on the LK63 human pre-B-cell line and overexpression in leukemic specimens and cell lines suggested a role in oncogenesis. We developed a biosensor-based approach using the immobilized HEK receptor exodomain to detect and monitor purification of the HEK ligand. A protein purification protocol, which included HEK affinity chromatography, achieved a 1.8 X 10(6)-fold purification of an approximately 23-kDa protein from human placental conditioned medium. Analysis of specific sHEK (soluble extracellular domain of HEK) ligand interactions in the first and final purification steps suggested a ligand concentration of 40 pM in the source material and a Kd of 2-3 nM. Since the purified ligand was N-terminally blocked, we generated tryptic peptides and N-terminal amino acid sequence analysis of 7 tryptic fragments of the S-pyridylethylated protein unequivocally matched the sequence for AL-1, a recently reported ligand for the related EPH-like RTK REK7 (Winslow, J.W., Moran, P., Valverde, J., Shih, A., Yuan, J.Q., Wong, S.C., Tsai, S.P., Goddard, A., Henzel, W.J., Hefti, F., Beck, K.D., & Caras, I.W. (1995) Neuron 14, 973-981). Our findings demonstrate the application of biosensor technology in ligand purification and show that AL-1, as has been found for other ligands of the EPH-like RTK family, binds more than one receptor.

Quantitation of the water channel protein aquaporin (CHIP28) from red blood cell membranes by densitometry of silver stained polyacrylamide gels.

A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.

A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system.

The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant (Robillard et al., 1996). The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I-. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation). The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991). When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30.

Expression cloning in Escherichia coli and preparative isolation of the reductase coacting with chalcone synthase during the key step in the biosynthesis of soybean phytoalexins

The cDNA for the reductase involved in the biosynthesis of 6′-deoxychalcone (4, 2′, 4′-trihydroxychalcone), the first specific intermediate in the pathway to soybean phytoalexins, was cloned into the expression vector pKK233-2 and transformed into Escherichia coli. Using this source, about 5 mg of homogeneous reductase was isolated from 45 g of cells. The protein purification protocol differs completely from the scheme applied to soybean cell cultures. Size, N-terminal and specific enzyme activities were identical for the plant and E. coli protein. The pure protein is fairly stable, retaining 70% of initial activity after storage at 5 °C during 4 weeks. This protein is used for crystallization and in the study of its protein-protein interaction with chalcone synthase.

Affinity chromatography on triazine dyes immobilized on novel perfluorocarbon supports

A bis-substituted conjugate of the dichlorotriazinyl dye C.I. Reactive Blue 4 (Procion Blue MX-R) with 1H,1H-pentadecafluorooctylamine was synthesised and characterised. This novel conjugate showed strong surface activity on aqueous-fluorocarbon interfaces and was used to prepare dyed liquid and solid perfluorocarbon supports for affinity chromatography. Rabbit muscle lactate dehydrogenase was purified up to 8.4-fold from a crude extract by chromatography on the surface active dye adsorbed on perfluorodecalin emulsion droplets and on a powdered microparticulate fluoropolymer. The performance of these novel affinity supports was shown to be comparable to adsorbents prepared by using the same triazine dye immobilised on agarose gels. Two further surface active dichlorotriazinyl dye conjugates were immobilised on liquid and solid perfluorocarbon supports and tested using protein purification protocols to illustrate the applicability of this novel technique.

Develop your next protein purification protocol in days, not weeks.

Develop your next protein purification protocol in days, not weeks.