Protease Recognition Sequence Research Articles

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors

BackgroundThe solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC).ResultsA single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods.ConclusionThe experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

The solubility and stability of recombinant proteins are increased by their fusion to NusA

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13–20 mg of fusion protein per litre of culture and 2.5–5 mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.

Mutations Leading to X-linked Hypohidrotic Ectodermal Dysplasia Affect Three Major Functional Domains in the Tumor Necrosis Factor Family Member Ectodysplasin-A

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.

Novel functional assay for proteases and modulators. Application in beta-secretase studies.

Proteases play a critical role in many cellular functions and have been an attractive therapeutic target due to their involvement in a number of disease processes. One prominent example is the secretases responsible for the generation of amyloid beta peptide, which is believed to be central for the development of Alzheimer's disease. It is therefore desirable to identify and characterize these proteases. We have developed a novel functional approach for identification of proteases and modulators by coupling the protease activity to caspase-mediated apoptosis. Here we show the proof of principle for this approach using beta-secretase as an example. We provide data showing that 1. A modified caspase-3 containing beta-secretase cleavage site induces apoptosis in 293T cells. 2. The modified caspase-3 induced apoptosis is correlated with the susceptibility of beta-secretase recognition sequence to beta-secretase. 3. In vivo beta-secretase competitors BACE2 and BACE2(D110A) prevent the modified caspase-3 induced cell death. Therefore, this approach can be a useful tool in studies of proteolytic cleavage provided only that the protease recognition sequence is known.

The furin protease cleavage recognition sequence of Sindbis virus PE2 can mediate virion attachment to cell surface heparan sulfate.

Cell culture-adapted Sindbis virus strains attach to heparan sulfate (HS) receptors during infection of cultured cells (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). At least three E2 glycoprotein mutations (E2 Arg 1, E2 Lys 70, and E2 Arg 114) can independently confer HS attachment in the background of the consensus sequence Sindbis virus (TR339). In the studies reported here, we have investigated the mechanism by which the E2 Arg 1 mutation confers HS-dependent binding. Substitution of Arg for Ser at E2 1 resulted in a significant reduction in the efficiency of PE2 cleavage, yielding virus particles containing a mixture of PE2 and mature E2. Presence of PE2 was associated with an increase in HS-dependent attachment to cells and efficient attachment to heparin-agarose beads, presumably because the furin recognition site for PE2 cleavage also represents a candidate HS binding sequence. A comparison of mutants with partially or completely inhibited PE2 cleavage demonstrated that efficiency of cell binding was correlated with the amount of PE2 in virus particles. Viruses rendered cleavage defective due to deletions of portions or all of the furin cleavage sequence attached very poorly to cells, indicating that an intact furin cleavage sequence was specifically required for PE2-mediated attachment to cells. In contrast, a virus containing a partial deletion was capable of efficient binding to heparin-agarose beads, suggesting different requirements for heparin bead and cell surface HS binding. Furthermore, virus produced in C6/36 mosquito cells, which cleave PE2 more efficiently than BHK cells, exhibited a reduction in cell attachment efficiency correlated with reduced content of PE2 in particles. Taken together, these results strongly argue that the XBXBBX (B, basic; X, hydrophobic) furin protease recognition sequence of PE2 can mediate the binding of PE2-containing Sindbis viruses to HS. This sequence is very similar to an XBBXBX heparin-HS interaction consensus sequence. The attachment of furin protease cleavage sequences to HS may have relevance to other viruses whose attachment proteins are cleaved during maturation at positively charged recognition sequences.

Identification of Protein-Protein Contacts between α/β-Type Small, Acid-soluble Spore Proteins of Bacillus Species Bound to DNA

Small, acid-soluble spore proteins (SASP) of the alpha/beta-type from several Bacillus species were cross-linked into homodimers, heterodimers and homooligomers with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of linear plasmid DNA. Significant protein cross-linking was not detected in the absence of DNA. In all four alpha/beta-type SASP examined, the amino donor in the EDC induced amide cross-links was the alpha-amino group of the protein. However, the carboxylate containing amino acid residues involved in cross-linking varied. In SASP-A and SASP-C of Bacillus megaterium two conserved glutamate residues, which form part of the germination protease recognition sequence, were involved in cross-link formation. In SspC from Bacillus subtilis and Bce1 from Bacillus cereus the acidic residues involved in cross-link formation were not in the protease recognition sequence, but at a site closer to the N terminus of the proteins. These data indicate that, although there are likely to be subtle structural differences between different alpha/beta-type SASP, the N-terminal regions of these proteins are involved in protein-protein interactions while in the DNA bound state.

Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.

Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.

A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.

Proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for its fusion activity.

The surface glycoprotein (S) of the murine hepatitis coronavirus MHV normally undergoes proteolytic cleavage during transport to the cell surface. To determine whether the cleavage of the MHV-JHM S glycoprotein is required to activate its ability to fuse cellular membranes, the protease recognition sequence in a cDNA copy of the S gene was altered from Arg-Arg-Ala-Arg-Arg into Ser-Val-Ser-Gly-Gly by site directed mutagenesis. The mutated and wild type S genes were expressed by means of recombinant vaccinia viruses and it could be shown that the mutated S protein was not cleaved when it was expressed in mouse DBT cells, in contrast to the wild type S protein. Nevertheless, the non-cleaved S protein induced extensive syncytium formation in mouse DBT cells. These results clearly indicate that the non-cleaved form of the MHV S protein is able to mediate cell membrane fusion. Thus, proteolytic cleavage is not an absolute requirement for its fusion function.

Genetically-Engineered Protease-Activated Triggers in a Pore-Forming Protein

ABSTRACTProtease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.

Genetically engineered bacteria to identify and produce anti-viral agents.

We have prepared a strain of Escherichia coli that expresses both the HIV protease and a Tet protein which has been modified to contain the HIV protease recognition sequence. When the protease is expressed, the bacteria will not grow in the presence of tetracycline. However, when the protease is inhibited the bacteria can grow in tetracycline containing media (Block and Grafstrom 1990). We have selected spontaneously arising Tet resistant mutants and have screened them for those that could be producing an inhibitor of HIV protease. The problems in the construction of this strain and the characterization of the various Tetr mutants are discussed.

Cleavage of the bovine herpesvirus glycoprotein B is not essential for its function.

Herpes simplex virus glycoprotein B (HSVgB) and its bovine herpesvirus homologue (BHVgB) share similar primary structures. These glycoproteins are present in the envelope of the virion and are believed to initiate infection by fusing the virus envelope with a host cell membrane. BHVgB, like the membrane-fusing glycoproteins of most enveloped viruses, is normally cleaved and is present as a disulphide-linked complex in the virus envelope and host cell membranes. HSVgB, however, remains uncleaved, presumably because it lacks a similar protease recognition sequence. To determine whether the cleavage of BHVgB is essential for its role in initiating infection, we altered the coding sequence of this glycoprotein by removing the protease cleavage site and making this region similar to that of HSVgB. The mutant BHVgB gene was expressed by an HSV recombinant virus in mouse L cells and produced an uncleaved BHVgB. The uncleaved BHVgB could complement the function of HSVgB which had been neutralized by monoclonal antibody H233. When expressed in mouse L cells, the uncleaved mutant BHVgB retained its ability to fuse membranes.

Novel bacteriological assay for detection of potential antiviral agents

A prototype assay for the initial screening of potential antiviral agents that uses bacterial growth on selective media is described. The human immunodeficiency virus (HIV) protease recognition sequence was inserted into the tetracycline resistance (Tet) protein encoded by plasmid pBR322 of Escherichia coli. Expression of both the HIV protease and the modified Tet protein prevented growth in the presence of tetracycline. However, inhibition of the HIV protease restored tetracycline resistance. Thus, potential HIV protease inhibitors can be identified by their ability to confer tetracycline resistance to this bacterial strain. The assay is simple, rapid, and inexpensive, and this concept can be applied to the search for inhibitors of other viral proteases.

Detection of the trans activity of the plum pox virus NIa-like protease in infected plants

The NIa-like protein of plum pox virus is a protease with high sequence specificity that is autocatalytically released from the viral polyprotein. In order to determine whether the protease is active in trans we constructed a fusion protein consisting of the C-terminal region of the plum pox virus polyprotein and the staphylococcal Protein A. The authentic protease recognition sequence Asn-Val-Val-Val-His-Gln-Ala occurs in the centre of this protein fusion. This protein was cleaved specifically by extracts of plum pox virus-infected plants due to the strong activity of the viral protease making it a useful tool for diagnostic purposes.

The complete nucleotide sequence of plum pox potyvirus RNA

The complete nucleotide sequence of the plum pox virus (PPV) RNA genome has been determined. The RNA sequence is 9786 nucleotides in length, excluding the 3′-terminal poly(A) tail. An AUG triplet at position 147–149 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3140 amino acid residues. The nucleotide sequence of the non-coding regions and the predicted amino acid sequence of the polyprotein of PPV were compared with those previously reported for two other potyviruses (tobacco etch virus, TEV, and tobacco vein mottling virus, TVMV), with nucleotide and amino acid sequences of other viruses, as well as with sequences from data banks. The potyvirus genomic expression is discussed in relation to the homologies observed, in particular the predicted protease recognition sequences in related viruses.