Henry Paulus

SINGLE-COLUMN PURIFICATION OF FREE RECOMBINANT PROTEINS USING A SELF-CLEAVABLE AFFINITY TAG DERIVED FROM A PROTEIN SPLICING ELEMENT

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

A rapid and sensitive method for measuring the binding of radioactive ligands to proteins

Abstract A new method is described for measuring the binding of radioactive ligands to proteins. The procedure involves the filtering of a solution of the protein and radioactive ligand through a Diaflo ultrafiltration membrane in a specially designed ultrafiltration cell. The membrane, on which the complex of protein and bound ligand is deposited, is then counted in a liquid scintillation spectrometer to yield directly the amounts of ligand bound. The ultrafiltration method of measuring ligand binding is sensitive and rapid. Binding studies can be carried out with microgram quantities of protein and at ligand concentrations exceeding the concentration of protein by several orders of magnitude. With the ultrafiltration cell described, eight samples can be processed simultaneously in less than an hour.

Protein splicing and autoproteolysis mechanisms

It has generally been assumed that the conversion of all inactive protein precursors to biologically active proteins is mediated by specific processing enzymes. However, numerous examples of self-catalyzed protein rearrangements have recently been discovered, including protein splicing and autoproteolysis of hedgehog proteins, glycosylasparaginases and pyruvoyl enzyme precursors. The initial formation of an ester bond by the acyl rearrangement of a peptide bond is a common feature of all of these autoprocessing reactions, which manifest themselves in diverse biological functions, which manifest themselves in diverse biological functions ranging from protein splicing to protein targeting, proenzyme activation, and the generation of enzyme-bound prosthetic groups. Although such acyl rearrangements are thermodynamically unfavorable, their coupling to diverse types of self-catalyzed irreversible steps drives the protein rearrangements to completion.

Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments.

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.

Protein Splicing in Vitro with a Semisynthetic Two-component Minimal Intein

Protein splicing elements, or inteins, catalyze their own excision from flanking polypeptide sequences, or exteins, thereby leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. A trans-splicing system, using separately purified and expressed N- and C-terminal intein fragments of about 100 amino acids each, fused to appropriate exteins, was recently derived from the Mycobacterium tuberculosis RecA intein (Mills, K. V., Lew, B. M., Jiang, S.-Q., and Paulus, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3543–3548). We have replaced the C-terminal intein fragment of this system with synthetic peptides comprising 35–50 of the C-terminal residues of the RecA intein. The N-terminal intein fragment and the synthetic peptide were reconstituted by renaturation from guanidinium chloride. In the absence of added reductants, a disulfide-linked dimer of the N-terminal fragment and the peptide accumulated and could be induced to splice by reduction of its disulfide bond. The intermediate and spliced products were identified by polyacrylamide gel electrophoresis, mass spectrometry, and derivatization with thiol-reactive biotin followed by Western blotting with a streptavidin-enzyme conjugate. This is the first example of protein splicing involving a synthetic intein fragment and opens the way for studying the active site structure and function of the intein by the use of different synthetic peptides, including ones with non-natural amino acids.

The enzymic formation of O-acetylhomoserine in Bacillus subtilis and its regulation by methionine and S-adenosylmethione

Abstract Cell-free extracts of Bacillus subtilis catalyze the formation of O-acetylhomoserine from acetyl-CoA and L-homoserine, while a corresponding reaction with succinyl-CoA is not observed. The formation of O-acetylhomoserine is subject to multivalent feedback inhibition by L-methionine and S-adenosylmethionine, implicating it as a step in methionine biosynthesis. Thus, in Bacilli the biosynthesis of both lysine and methionine appears to involve acetylated intermediates, in contrast to Escherichia coli where the corresponding succinylated derivatives are used.

Characteristics of protein splicing in trans mediated by a semisynthetic split intein

Protein splicing in trans results in the ligation of two protein or peptide segments linked to appropriate intein fragments. We have characterized the trans-splicing reaction mediated by a naturally expressed, approximately 100-residue N-terminal fragment of the Mycobacterium tuberculosis intein and a synthetic peptide containing the 38 C-terminal intein residues, and found that the splicing reaction was very versatile and robust. The efficiency of splicing was nearly independent of temperature between 4 and 37 degrees C and pH between 6.0 and 7.5, with only a slight decline at pH values as high as 8.5. In addition, there was considerable flexibility in the choice of the C-terminal intein fragment, no significant difference in protein ligation efficiency being observed between reactions utilizing the N-terminal fragment and either the naturally expressed 107-residue C-terminal portion of the intein, much smaller synthetic peptides, or the 107-residue C-terminal intein fragment modified by fusion of a maltose binding protein domain to its N-terminus. The ability to use different types of the C-terminal intein fragments and a broad range of reaction conditions make protein splicing in trans a versatile tool for protein ligation.

Determination of dipicolinic acid by high-pressure liquid chromatography

Abstract A method for the analysis of dipicolinic acid by high-pressure liquid chromatography on a weak anion exchanger in the presence of an EDTA buffer is described. The method requires only simple sample processing, is rapid, and provides a linear response to dipicolinate concentration over a range of 0.02 to 10 μg. The dipicolinate content of less than 1 μg of bacterial spores can be measured and the presence of nonsporulatd bacterial cells causes no interference.

An in vivo screening system against protein splicing useful for the isolation of non-splicing mutants or inhibitors of the RecA intein of Mycobacterium tuberculosis.

Protein splicing involves the self-catalyzed excision of an intervening sequence, the intein, from a precursor protein, with the concomitant ligation of the flanking extein sequences to yield a new polypeptide. The ability of inteins to promote protein splicing even when inserted into a foreign context has facilitated the study of the modulation of protein splicing. In this paper, we describe an in vivo screening system for the isolation of mutations or inhibitors that interfere with protein splicing mediated by the RecA intein of Mycobacterium tuberculosis. It involves the activation of the cytotoxic CcdB protein by protein splicing, such that host cells survive in the presence of inducer only when protein splicing is blocked. The coding sequence for the RecA intein was inserted in-frame into the polylinker region of an inducible lacZ alpha-ccdB fusion vector, leading to inactivation of the CcdB toxin unless the intein is excised by protein splicing. Depending on the objective of the screening procedure, its stringency can be modified by altering the level of expression of the intein-CcdB fusion protein. To induce large amounts of CcdB fusion proteins, the fusion protein is expressed from a high-copy-number plasmid. Such a screening system detects even low levels of protein splicing and we have used it to show that protein splicing of the RecA intein is compatible with any amino acid in the extein position adjacent to the N-terminal splice junction. In order to search for protein splicing inhibitors, which may attenuate protein splicing by less than an order of magnitude, we have also constructed a low-copy-number intein-CcdB plasmid so that the host cells can survive when splicing of the expressed CcdB fusion protein is only moderately suppressed. We anticipate that the CcdB-based in vivo screening system will find uses in the analysis of structural and mechanistic aspects of protein splicing.

Studies on ribonucleoside-diphosphate reductase in permeable animal cells: I. Reversible permeabilization of mouse L cells with dextran sulfate☆

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [3H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [3H]dTTP into DNA and for K+- and ATP-dependent incorporation of [3H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH2PO4, glucose, glutamine, and CaCl2, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.