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Thrombin, a major protein involved in the clotting cascade by the conversion of inactive fibrinogen to fibrin, plays a crucial role in the development of thrombosis. Antithrombin nanoparticles enable site-specific anticoagulation without increasing bleeding risk. Here we outline the process of making and the characterization of bivalirudin and D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK) nanoparticles. Additionally, the characterization of these nanoparticles, including particle size, zeta potential, and quantification of PPACK/bivalirudin loading, is also described.

The mechanisms behind the Amyloid-β (Aβ) peptide neurotoxicity in Alzheimer's disease are intensely studied and under debate. One suggested mechanism is that the peptides assemble in biological membranes to form β-barrel shaped oligomeric pores that induce cell leakage. Direct detection of such putative assemblies and their exact oligomeric states is however complicated by a high level of heterogeneity. The theory consequently remains controversial, and the actual formation of pore structures is disputed. We herein overcome the heterogeneity problem by employing a native mass spectrometry approach and demonstrate that Aβ(1-42) peptides form coclusters with membrane mimetic detergent micelles. The coclusters are gently ionized using nanoelectrospray and transferred into the mass spectrometer where the detergent molecules are stripped away using collisional activation. We show that Aβ(1-42) indeed oligomerizes over time in the micellar environment, forming hexamers with collision cross sections in agreement with a general β-barrel structure. We also show that such oligomers are maintained and even stabilized by addition of lipids. Aβ(1-40) on the other hand form significantly lower amounts of oligomers, which are also of lower oligomeric state compared to Aβ(1-42) oligomers. Our results thus support the oligomeric pore hypothesis as one important cell toxicity mechanism in Alzheimer's disease. The presented native mass spectrometry approach is a promising way to study such potentially very neurotoxic species and how they could be stabilized or destabilized by molecules of cellular or therapeutic relevance.

Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

MMP9-responsive bivalirudin-HPMA copolymers were synthesized for direct, local administration in rat spinal cord contusion injury models. Polymer-conjugated bivalirudin peptides maintained activity while demonstrating enzyme-mediated release upon MMP9 exposure and prolonged release from hyaluronic acid/methylcellulose (HAMC) hydrogels compared to free bivalirudin peptide. Localized administration of bivalirudin copolymers in vivo at the site of rat spinal cord injury decreased cellular proliferation and astrogliosis, suggesting the bivalirudin copolymer and HAMC hydrogel system are a promising therapeutic intervention for reducing immediate inflammatory responses and long term scarring.

The RGD sequence was used to design potent hirudin isoform 3 mimetic peptides with both antithrombin activity and antiplatelet aggregation activity. The RGD and proline were inserted between the catalytic active binding domain (D-Phe-Pro-Arg-Pro) on the N-terminus and the anion-binding exosite binding domain (QGDFEPIPEDAYDE) on the Cterminus. Thrombin titration assay and ATP-induced platelet aggregation test revealed that the peptide with the linker RGDWP or RGDGP possessed potent antithrombin and antiplatelet activities, while other peptides without the Pro residue in the linker only showed antithrombin activity. Similar results were obtained in the RGD-containing hirulog-1 variants. Our study indicates that the inserted Pro residue facilitates the exposure of RGD and the binding of the peptide to glycoprotein IIb/IIIa (GPIIb/IIIa). The strategy of combining the RGD sequence and the Pro residue may be used for future designs of bifunctional antithrombotic agents.

Amelogenin plays a key role in the formation of the highly mineralized structure of tooth enamel. During the secretory stage, amelogenin is cleaved gradually by a protease, matrix metalloproteinase-20 (MMP-20), releasing hydrophilic C-terminal peptides. In this study, the biophysical properties of synthetic C-terminal peptides (of 28, 17, and 11 residues), mimicking native peptides, were explored in vitro. A sudden decrease was observed in the zeta (ζ)-potential upon the addition of calcium or phosphates, which was also accompanied by an increased aggregation propensity of the peptides. Under most of the experimental conditions, the particle size increased at a pH 2-3 units higher than the isoelectric point (pI) of the peptides, while the peptides existed as smaller particles (<2 nm) near their pI values and in the acidic range. They showed poor affinity for calcium and phosphates, comparable to full-length amelogenin and variants. The secondary structure determination showed that the 11-amino-acid peptide contained defined secondary structure comprising beta-sheets and turns. Atomic force microscopy analysis revealed the presence of thin, disk-like nanostructures of 54.4 nm diameter for the 28-amino-acid peptide and 54.9 nm diameter for the 11-amino acid peptide, whereas no definite structures were observed for the 17-amino-acid peptide. It is concluded that the amelogenin C-terminal peptides are capable of interacting with calcium and phosphate ions, of self-assembly into nanostructures, and may have some secondary structure, and hence may have some role in enamel synthesis., (© 2012 Eur J Oral Sci.)

In order to determine whether the Ii-Key technology can enhance the presentation of specific epitopes associated with type 1 diabetes, we have designed and synthesized a series of Ii-Key/proinsulin and GAD epitope hybrid peptides. Peptides of proinsulin and GAD shown to be recognized by CD4+ T cells of type 1 diabetes patients have been selected from the literature and modified with Ii-Key. A total of 23 Caucasian type 1 diabetes subjects and 17 normal subjects as controls were included in the study. Reactive T cells were identified using an IFN-γ ELISPOT assay. We selected 5 proinsulin and 5 GAD epitopes. Regarding the activity of the proinsulin Ii-Key hybrids, 3 out of 15 patients (20%) demonstrated a positive response to one or more Ii-Key hybrid peptides compared to no responders in the control subjects. Two out of 8 patients demonstrated a positive response to one or more Ii-Key/GAD65 hybrids. Proinsulin Ii-Key hybrids and peptides were recognized only by DR3/DR4 0302+ve diabetic patients. Control subjects showed no detectable response to stimulation with Ii-Key hybrids or peptides, neither for proinsulin nor GAD65. We have now shown that the use of Ii-Key-modified MHC class II epitopes, derived from proteins associated with insulin-secreting cells, can detect the presence of specifically activated CD4+ T helper cells with greater sensitivity than unmodified epitopes in the standard ELISPOT assay. The use of these technologies may be of use in identifying patients at the earliest stages of type 1 diabetes., (© Georg Thieme Verlag KG Stuttgart · New York.)

In the present study, we investigated the metal-binding reactivity of the isolated alpha domain of human metallothionein isoform 1a, with specific emphasis on resolving the debate concerning the cooperative nature of the metal-binding mechanism. The metallation reaction of the metal-free alpha domain with Cd2+ was unequivocally shown to proceed by a noncooperative mechanism at physiologic pH by CD and UV absorption spectroscopy and ESI MS. The data clearly show the presence of intermediate partially metallated metallothionein species under limiting Cd2+ conditions. Titration with four molar equivalents of Cd2+ was required for the formation of the Cd4alpha species in 100% abundance. The implications of a noncooperative metal-binding mechanism are that the partially metallated and metal-free species are stable intermediates, and thus may have a potential role in the currently undefined function of metallothionein.

There are many precedents for the production from a single precursor of multiple peptides, with independent receptors and different bioactivities. Gastrin-releasing peptide (GRP) is initially synthesized as amino acids 1-27 of a 125-residue precursor, proGRP, and is subsequently cleaved and amidated to form GRP18-27. We investigated the hypothesis that C-terminal proGRP peptides are also biologically active. Human proGRP18-125 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Recombinant proGRP18-125 stimulated proliferation and migration of the human colorectal carcinoma cell line DLD-1. The observations that an antagonist selective for the GRP receptor did not inhibit activity in either proliferation or migration assays and that the recombinant peptide did not bind to either the GRP receptor or orphan receptor BRS-3 indicated that neither activity was mediated by the known GRP receptors. Recombinant human proGRP31-125 and proGRP42-98 were also prepared and shown to stimulate proliferation of DLD-1 cells and the human prostate carcinoma cell line DU145. The synthetic peptides proGRP47-68 and [Tyr79]proGRP80-97 stimulated inositol phosphate production, MAPK kinase activity, and proliferation and migration of DLD-1 cells. Binding sites for both radioiodinated synthetic peptides were demonstrated on DLD-1 cells. Each peptide was able to compete with the other for binding, and a GRP receptor antagonist did not inhibit binding of either peptide. We conclude that peptides derived from the C terminus of proGRP are biologically active and that their activity is mediated by a receptor distinct from the two known GRP receptors.

Synthetic peptides have been shown to evoke neutralizing and cytotoxic protective anti-HIV responses in mice and other animal models. Recent data support that C4V3 peptides can induce anti- V3 antibodies that neutralize primary isolates. Critical to the success of peptide-based vaccines is the development of strategies to augment their immunogenicity while reducing their large-scale production costs. Therefore, finding efficient and economical alternatives for the production of epitopic vaccines could have an impact on researches using such immunogens. Herein, we report the recombinant production and immunological characterization of a short polypeptide which carries the three relevant epitopes contained in a C4V3 peptide. This polypeptide, named rC4V3, was efficiently produced in E. coli, yielding more than 75 mg per culture liter. No major difficulties were found in the recovery, refolding and purification of this peptide; the latter facilitated by C-terminal inclusion of a histidine tag. The immunogenicity of this protein was studied by administering it intramuscularly or intranasally to mice and it demonstrated to be a strong elicitor of anti-HIV antibodies at systemic and mucosal compartments. Remarkably, such responses were attained with rC4V3 even without the need of adjuvants. We can conclude that this protein might be a promising tool for studies using epitope-based vaccine designs.

Measuring the interaction of class I human leukocyte antigens (HLA) and their peptide epitopes acts as a guide for the development of vaccines, diagnostics, and immune-based therapies. Here, we report the development of a sensitive biochemical assay that relies upon fluorescence polarization to indicate peptide interactions with recombinant soluble HLA proteins. It is a cell- and radioisotope-free assay that has the advantage of allowing the direct, real-time measurement of the ratio between free and bound peptide ligand in solution without separation steps. Peptide/HLA assay parameters were established using several HLA A*0201-specific fluorescein isothiocyanate-labeled peptides. Optimal loading of synthetic peptides into fully assembled soluble HLA-A*0201 complexes was enabled by thermal destabilization at 53 degrees C for 15 min, demonstrating that efficient peptide exchange does not require the removal of endogenous peptides from the reaction environment. An optimal ratio of three beta-2 microglobulin molecules per single HLA heavy chain was determined to maximize peptide binding. Kinetic binding studies indicate that soluble HLA-A*0201/peptide interactions are characterized by a range of moderate k(on) values (1 x 10(4) to 8.7 x 10(4) M(-1) s(-1)) and slow k(off) values (1.9 x 10(-4) to 4.3 x 10(-4) s(-1)), consistent with parameters for native HLA molecules. Testing of the A*0201-specific peptides with 48 additional class I molecules demonstrates that the unique peptide binding behavior of individual HLA molecules is maintained in the assay. This assay therefore represents a versatile tool for characterizing the binding of peptide epitopes during the development of class I HLA-based vaccines and immune therapies.

Two recombinant collagen-like proteins consisting of cell adhesion domains derived from native type I collagen were designed and synthesized by a genetic engineering method. The cross-linking sequence, GPPGPCCGGG, derived from collagen III was used to promote triple helix formation through the disulfide bonds formed among three chains by flanking the peptide at the C-terminal of the collagen-like proteins. SDS-PAGE and western-blotting data suggested possibility of the formation of a triple helix structure for both recombinant proteins. CD spectra and thermal stability analyses indicated that the triple-helix structure in the collagen-like proteins was pH-dependent and stabilized under acidic environmental condition. Moreover, the collagen-like protein flanked with the cross-linking sequence at the C-terminal showed the most stable triple-helical conformation under acidic conditions.

A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties. A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential. Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers. We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al. Nat. Biotechnol, 2000, 18:194-198), as a reference peptide. We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17. Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells. The proliferation inhibition shown by V2 was slightly better than that shown by V1. Recombinant peptides V2 and V3 were produced on a large scale in an E. coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30%. Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E. coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells. Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.

To follow Mg2+ binding to the N-terminal of calmodulin (CaM), we substituted Phe in position 19, which immediately precedes the first Ca2+/Mg2+ binding loop, with Trp, thus making F19WCaM (W-Z). W-Z has four acidic residues in chelating positions, two of which form a native Z-acid pair. We then generated seven additional N-terminal CaM mutants to examine the role of chelating acidic residues in Mg2+ binding and exchange with the first EF-hand of CaM. A CaM mutant with acidic residues in all of the chelating positions exhibited Mg2+ affinity similar to that of W-Z. Only CaM mutants that had a Z-acid pair were able to bind Mg2+ with physiologically relevant affinities. Removal of the Z-acid pair from the first EF-hand produced a dramatic 58-fold decrease in its Mg2+ affinity. Additionally, removal of the Z-acid pair led to a 1.8-fold increase in the rate of Mg2+ dissociation. Addition of an X- or Y-acid pair could not restore the high Mg2+ binding lost with removal of the Z-acid pair. Therefore, the Z-acid pair in the first EF-hand of CaM supports high Mg2+ binding primarily by increasing the rate of Mg2+ association.

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

To enable metal affinity purification of cytochrome c oxidase reconstituted into phospholipid vesicles, a histidine-tag was engineered onto the C-terminal end of the Rhodobacter sphaeroides cytochrome c oxidase subunit II. Characterization of the natively processed wildtype oxidase and artificially processed forms (truncated with and without a his-tag) reveals Km values for cytochrome c that are 6-14-fold higher for the truncated and his-tagged forms than for the wildtype. This lowered ability to bind cytochrome c indicates a previously undetected role for the C-terminus in cytochrome c binding and is mimicked by reduced affinity for an FPLC anion exchange column. The elution profiles and kinetics indicate that the removal of 16 amino acids from the C-terminus, predicted from the known processing site of the Paracoccus denitrificans oxidase, does not produce the same enzyme as the native processing reaction. MALDI-TOF MS data show the true C-terminus of subunit II is at serine 290, three amino acids longer than expected. When the his-tagged form is reconstituted into lipid vesicles and further purified by metal affinity chromatography, significant improvement is observed in proton pumping analysis by the stopped-flow method. The improved kinetic results are attributed to a homogeneous, correctly oriented vesicle population with higher activity and less buffering from extraneous lipids.

Protein kinase CK2 ("casein kinase 2") holoenzyme is composed of two catalytic (alpha and/or alpha') and two regulatory beta-subunits. A truncated form of the beta-subunit lacking its C-terminal region (betaDelta171-215) has lost the ability to stably associate with the catalytic subunits and to display a number of properties which are mediated by structural elements still present in its sequence, notably down-regulation of catalytic activity, autophosphorylation, and responsiveness to polycationic effectors. All these functions are restored by simultaneous addition of a synthetic peptide reproducing the deleted fragment, beta170-215, which is able to associate with the catalytic subunits and to stimulate catalytic activity. This peptide includes a segment displaying significant sequence similarity with a region of cyclin A which interacts with the PSTAIRE motif of CDK2 eliciting its catalytic activity. A peptide reproducing this sequence (beta181-203), but not its derivative in which three nonpolar side chains have been replaced by polar ones, interacts with the alpha-subunit and stimulates its catalytic activity; it also partially restores the ability of truncated betaDelta171-215 to autophosphorylate. These data disclose the essential role of a structural module located between residues 181 and 203 in conferring regulatory properties to the beta-subunit of CK2.

The insulin-responsive glucose transporter, Glut4, exhibits a unique subcellular distribution such that in the absence of insulin >95% of the protein is stored within intracellular membranes. In response to insulin, Glut4 exhibits a large mobilisation to the plasma membrane. Studies of the amino acid motifs which regulate the unique trafficking of Glut4 have identified several key residues within the soluble cytoplasmic N- and C-terminal domains of Glut4. Of particular note is a Leu-498Leu-499 motif within the C-terminal domain that has been proposed to regulate both internalisation from the plasma membrane and sorting to an insulin-sensitive compartment. In this study, we have examined the role of the adjacent amino acids (Glu-491, Gln-492 and Glu-493) by their sequential replacement with Ala. Our results are consistent with the notion that Glu-491 and Glu-493 play an important role in the sub-endosomal trafficking of Glut4, as substitution of these residues with Ala results in increased levels of these proteins at the cell surface, reduced insulin-stimulated translocation and increased susceptibility to endosomal ablation. These residues, together with other identified sequences within the C-terminus of Glut4, are likely to be crucial targeting elements that regulate Glut4 subcellular distribution.

The nicotinamide adenine dinucleotide phosphate (NADPH) binding site of the NADPH oxidase complex is believed to be located on the beta, subunit of cytochrome b558. However, our previous studies showed that p67PHOX also contains an NADPH binding site that is essential for normal oxidase activity and that p67PHOX is able to mediate a slow electron transfer from a reduced pyridine nucleotide to an artificial electron acceptor. Using both affinity labeling and fluorescence quenching, we have obtained further evidence that p67PHOX is able to bind NADPH. We have used a number of truncated forms of p67PHOX, including p67PHOX(1-243), p67PHOX(1-210), p67PHOX(1-199), and p67PHOX(244-526) (where the numbers represent the initial and final amino acids in the truncated p67PHOX) in order to localize the binding site. We found that NADPH could bind to p67PHOX(1-243), p67PHOX(1-210), and p67PHOX(1-199) but not to p67PHOX(244-526). The p67PHOX(1-199) fragment consists largely of four tetratricopeptide (TPR) domains. We showed further that Rac2-GTP gamma S and to a lesser extent Rac2-GDP beta S could modulate the binding of NADPH to p67PHOX.

In vitro folding of mature subtilisin is extremely slow. The isolated pro-domain greatly accelerates in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In our studies of subtilisin, we are trying to answer two basic questions: why does subtilisin fold slowly without the pro-domain and what does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants governing individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro folding experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular folding reaction of subtilisin and proR9 occurs in two steps: an initial binding of proR9 to unfolded subtilisin, followed by isomerization of the initial complex into the native complex. The central findings are as follows. First, the independently stable proR9 folds subtilisin much faster than the predominantly unfolded wild-type pro-domain. Second, at micromolar concentrations of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native conformation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin, which form the initial complex with proR9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the native form slowly and the remaining 23% isomerizes more rapidly (1.5 s-1). The slow-folding population may be unfolded subtilisin with the trans form of proline 168, which must isomerize to the cis form during refolding. Third, in the absence of proline isomerization, the rate of subtilisin folding is rapid and at [proR9] 3 s-1. The implications of these results concerning why subtilisin folds slowly without the pro-domain are discussed.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.

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., (Copyright 2000 John Wiley & Sons, Inc.)

The specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D-FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests. r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D-FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.

An intact gene coding for wild type diphtheria toxin was reconstituted in vitro from fragments of cloned inactive diphtheria toxin mutants with defects in different parts of the gene. The reconstituted DNA template was amplified using the Taq DNA polymerase chain reaction, providing a virtually unlimited supply. The toxin was expressed in vitro by transcription from a T3 RNA polymerase promoter, followed by translation of the mRNA in a rabbit reticulocyte lysate system. Diphtheria toxin synthesised in this manner was shown to be as toxic as natural toxin and behaved like natural toxin in every way tested. This can serve as a general approach for study of toxic proteins, opening up the possibility of in vitro gene manipulation. Reconstitution of the wild type gene from fragments followed by polymerase chain reaction amplification bypasses the hazards of cloning protein toxins.