A method for the prevention of thrombin-induced degradation of recombinant proteins

A new strategy to prevent degradation of recombinant proteins caused by non-specific cleavage by thrombin is described. We demonstrate that degradation due to non-specific cleavage of recombinant protein mediated by thrombin can be completely prevented by separation of thrombin from the recombinant protein on spin columns packed with heparin-sepharose. This method is generally applicable to all recombinant proteins that require the thrombin for the cleavage of affinity tags for purification. To our knowledge, this is the first report of an efficient and reliable method for the separation of residual thrombin from purified recombinant proteins.     

The Interaction between Factor H and Von Willebrand Factor

Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13.     

Molecular Characterization of Proteolytic Processing of the Gag Proteins of Human Spumavirus

Spumaviruses, or foamy viruses, express Gag proteins that are incompletely processed by the viral protease in cell cultures. To delineate the proteolytic cleavage sites between potential Gag subdomains, recombinant human spumaretrovirus (HSRV) Gag proteins of different lengths were expressed, purified by affinity chromatography, and subjected to HSRV protease assays. HSRV-specific proteolytic cleavage products were isolated and characterized by Western blotting. Peptides spanning potential cleavage sites, as deduced from the sizes of the proteolytic cleavage products, were chemically synthesized and assayed with HSRV protease. The cleaved peptides were then subjected to mass spectrometry. In control experiments, HSRV protease-deficient mutant proteins were used to rule out unspecific processing by nonviral proteases. The cleavage site junctions identified and the calculated sizes of the cleavage products were in agreement with those of the authentic cleavage products of the HSRV Gag proteins detectable in viral proteins from purified HSRV particles and in virus-infected cells. The biological significance of the data was confirmed by mutational analysis of the cleavage sites in a recombinant Gag protein and in the context of the infectious HSRV DNA provirus.     

Apoptotic cleavage of scaffold attachment factor A (SAF-A) by caspase-3 occurs at a noncanonical cleavage site

Members of the caspase family of cysteine proteases play essential roles in the disintegration of cellular architecture during apoptosis. Caspases have been grouped into subfamilies according to their preferred cleavage sites, with the "apoptotic executioner" caspase-3 as the prototype of DEXD-dependent proteases. We show here that caspase-3 is more tolerant to variations of the cleavage site than previously anticipated and present an example of a noncanonical recognition site that is efficiently cleaved by caspase-3 in vitro and in vivo. The new cleavage site was identified in human scaffold attachment factor A, one of the major scaffold attachment region DNA-binding proteins of human cells thought to be involved in nuclear architecture by fastening chromatin loops to a proteinaceous nuclear skeleton, the so-called nuclear matrix or scaffold. Using an amino-terminal recombinant construct of scaffold attachment factor A and recombinant caspase-3, we have mapped the cleavage site by matrix-assisted laser desorption ionization/time of flight mass spectrometry and Edman sequencing. We find that cleavage occurs after Asp-100 in a sequence context (SALD) that does not conform to the hitherto accepted DEXD consensus sequence of caspase-3. A point mutation, D100A, abrogates cleavage by recombinant caspase-3 in vitro and during apoptosis in vivo, confirming SALD as a novel caspase-3 cleavage site.     

The use of recombinant fusion proteases in the affinity purification of recombinant proteins

In the affinity purification of recombinant fusion proteins, the rate-limiting step is usually the efficient proteolytic cleavage and removal of the affinity tail and the protease from the purified recombinant protein. We have developed a rapid, convenient, and efficient method of affinity purification that can overcome this limitation. In one example of the method, the protease 3C from a picornavirus (3Cpro), which cleaves specific sequences containing a minimum of 6-7 amino acids, has been expressed as a fusion with glutathione S-transferase. The resultant recombinant "fusion protease" cleaves fusion proteins bearing (from the amino-terminus) the same affinity tail as the fusion protease, a 3Cpro cleavage recognition site, and the recombinant protein of interest. The recombinant protein is purified in a single chromatographic step, which removes both the affinity tail and the fusion protease. The advantages over existing methods include much improved specificity of proteolytic cleavage, complete removal of the protease and the affinity tail in one step, and the option of adding any desired amount of fusion protease to ensure efficient cleavage. The potential flexibility of the method is shown by the use of various affinity tails and alternative fusion proteases.     

Proteome-wide Identification of HtrA2/Omi Substrates

To identify apoptotic targets of HtrA2/Omi, we purified recombinant HtrA2/Omi and its catalytically inactive S306A mutant. Lysates of human Jurkat T lymphocytes incubated with either wild-type recombinant HtrA2/Omi or the S306A mutant were screened using the gel-free COFRADIC approach that isolates peptides covering the N-terminal parts of proteins. Analysis of the 1162 proteins identified by mass spectrometry yielded 15 HtrA2/Omi substrates of potential physiological relevance together holding a total of 50 cleavage sites. Several processing events were validated by incubating purified recombinant HtrA2/Omi with in vitro translated substrates or with Jurkat cell lysates. In addition, the generated set of cleavage sites was used to assess the protein substrate specificity of HtrA2/Omi. Our results suggest that HtrA2/Omi has a rather narrow cleavage site preference and that cytoskeletal proteins are prime targets of this protease.     

Study of protein splicing and intein-mediated peptide bond cleavage under high-cell-density conditions

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.     

Different Regions of the Newcastle Disease Virus Fusion Protein Modulate Pathogenicity

Newcastle disease virus (NDV), also designated as Avian paramyxovirus type 1 (APMV-1), is the causative agent of a notifiable disease of poultry but it exhibits different pathogenicity dependent on the virus strain. The molecular basis for this variability is not fully understood. The efficiency of activation of the fusion protein (F) is determined by presence or absence of a polybasic amino acid sequence at an internal proteolytic cleavage site which is a major determinant of NDV virulence. However, other determinants of pathogenicity must exist since APMV-1 of high (velogenic), intermediate (mesogenic) and low (lentogenic) virulence specify a polybasic F cleavage site. We aimed at elucidation of additional virulence determinants by constructing a recombinant virus that consists of a lentogenic NDV Clone 30 backbone and the F protein gene from a mesogenic pigeon paramyxovirus-1 (PPMV-1) isolate with an intracerebral pathogenicity index (ICPI) of 1.1 specifying the polybasic sequence R-R-K-K-R*F motif at the cleavage site. The resulting virus was characterized by an ICPI of 0.6, indicating a lentogenic pathotype. In contrast, alteration of the cleavage site G-R-Q-G-R*L of the lentogenic Clone 30 to R-R-K-K-R*F resulted in a recombinant virus with an ICPI of 1.36 which was higher than that of parental PPMV-1. Substitution of different regions of the F protein of Clone 30 by those of PPMV-1, while maintaining the polybasic amino acid sequence at the F cleavage site, resulted in recombinant viruses with ICPIs ranging from 0.59 to 1.36 suggesting that virulence is modulated by regions of the F protein other than the polybasic cleavage site.     

Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.     

Hamster Bcl-2 protein is cleaved in vitro and in cells by caspase-9 and caspase-3

Full-length cDNA of hamster bcl-2 (771 nt) was cloned by RT-PCR and inserted into pGEX-4T-1 to produce the recombinant hamster Bcl-2 protein. The purified recombinant Bcl-2 protein (26.4 kDa) was used as a substrate for the active human caspase-3 and caspase-9 in vitro. It is shown here that Bcl-2 is efficiently cleaved by caspase-3 to a 23 kDa fragment. Although not possessing a putative caspase-9 cleavage site in its sequence, hamster Bcl-2 was also cleaved by caspase-9 into exactly the same 23 kDa cleavage product, indicating that cleavage occurred at the same site. Caspase-3- and caspase-9-mediated cleavage of Bcl-2 was efficiently blocked by caspase-3 (zDEVD) and caspase-9 (zLEHD) inhibitor, respectively. We also show that caspase-9/-3-mediated cleavage of Bcl-2 occurs in vivo during apoptosis in CHO-HSV-TK cells after exposure to the antiviral drug ganciclovir.     

Strategy for improvement of enteropeptidase efficiency in tag removal processes

Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. It has also great biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occurred during cleavage of fusions when high amount of enzyme is required. In this study we have improved the efficiency of fusion proteins cleavage by enteropeptidase by substitution of the Lys residue by Arg in specific cleavage sequence (Asp)(4)-Lys. We have demonstrated that 3-6-fold lower amounts of the catalytic subunit of human and bovine enteropeptidase is required for 95% cleavage of Trx/TRAIL and Trx/FGF-2 fusions with (Asp)(4)-Arg cleavage sequence in comparison to native sequence (Asp)(4)-Lys. As a result, reduced amount of non-specifically cleaved peptide fragments were observed during cleavage of (Asp)(4)-Lys/Arg mutated fusions. These findings overcome limitations of enteropeptidase in tag removal processes during recombinant proteins purification and extend its commercial benefit in the biopharmaceutical industry.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.     

A faster migrating variant masquerades as NICD when performing in vitro gamma-secretase assays with bacterially expressed Notch substrates

Intramembrane proteolysis is a new and rapidly growing field. In vitro assays utilizing recombinant substrates for gamma-secretase, an intramembrane-cleaving enzyme, are critically important in order to characterize the biochemical properties of this unusual enzyme. Several recombinant Notch proteins of varying length are commonly used as in vitro substrates for CHAPSO-solubilized gamma-secretase. Here we report that several recombinant Notch constructs undergo limited or no proteolysis in vitro. Instead, upon incubation with or without gamma-secretase, variants of the intact protein migrate during SDS-PAGE at the location expected for the gamma-secretase specific cleavage products. In addition, we show that addition of aspartyl- and gamma-secretase specific protease inhibitors are able to retard the formation of these variants independent of gamma-secretase, which could lead to the erroneous conclusion that Notch cleavage by solubilized gamma-secretase was achieved in vitro even when no proteolysis occurred. In contrast, substrates produced in mammalian or insect cells are cleaved efficiently in vitro. These observations suggest that in vitro studies reliant on recombinant, bacterially produced Notch TMD should be performed with the inclusion of additional controls able to differentiate between actual cleavage and this potential artifact.     

Simple purification ofEscherichia coli-derived recombinant human interleukin-2 expressed with N-terminus fusion of glucagon

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed inEscherichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced inE. coli cytoplasm were easily dissolved by simple alkaline pH shift (8→12→8). Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.     

Recombinant RNase Z does not recognize CCA as part of the tRNA and its cleavage efficieny is influenced by acceptor stem length

One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3'-terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'-trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wild-type tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.     

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.     

Truncation of the amino-terminus of the recombinant aggrecan rAgg1mut leads to reduced cleavage at the aggrecanase site. Efficient aggrecanase catabolism may depend on multiple substrate interactions.

Aggrecanase cleavage at the Glu(373)-Ala(374) site in the interglobular domain of the cartilage proteoglycan aggrecan is a key event in arthritic diseases. The observation that substrates representing only the aggrecanase cleavage site are not catabolized efficiently by aggrecanase prompted us to investigate the requirement of aggrecanase for additional structural elements of its substrate other than the actual cleavage site. Based on the recombinant substrate rAgg1mut we constructed deletion mutants with successively truncated N- or C-termini of the interglobular domain. Catabolism by aggrecanase activities induced in rat chondrosarcoma cells, porcine chondrocytes, and by human recombinant ADAMTS4 showed a gradually decreasing catabolism of progressively shortened, N-terminal deletion mutants of the substrate rAgg1mut. A reduction to 32 amino acids N-terminal to the aggrecanase site resulted in a decrease of at least 42% of aggrecanase cleavage products as compared with the wild-type substrate. When only 16 amino acids preceded the Glu(373)-Ala(374) site, aggrecanase cleavage was completely inhibited. In contrast, C-terminal deletions did not negatively affect aggrecanase cleavage up to the reduction to 13 amino acids C-terminal to the cleavage site. Unlike aggrecanase(s), membrane type 1-matrix metalloprotease (MT1-MMP), able to cleave rAgg1mut both at the aggrecanase and the MMP site, was insensitive to N-terminal deletions regarding aggrecanase cleavage, indicating that the importance of the N-terminus is characteristic for aggrecanase(s). Taken together, the results demonstrate that the amino-terminus of rAgg1mut, containing the MMP site, plays an important role for efficient cleavage by aggrecanase(s), possibly by serving as a further site of interaction between the enzyme and its substrate.     

SLRP interaction can protect collagen fibrils from cleavage by collagenases

Decorin, fibromodulin and lumican are small leucine-rich repeat proteoglycans (SLRPs) which interact with the surface of collagen fibrils. Together with other molecules they form a coat on the fibril surface which could impede the access to collagenolytic proteinases. To address this hypothesis, fibrils of type I or type II collagen were formed in vitro and treated with either collagenase-1 (MMP1) or collagenase-3 (MMP13). The fibrils were either treated directly or following incubation in the presence of the recombinant SLRPs. The susceptibility of the uncoated and SLRP-coated fibrils to collagenase cleavage was assessed by SDS/PAGE. Interaction with either recombinant decorin, fibromodulin or lumican results in decreased collagenase cleavage of both fibril types. Thus SLRP interaction can help protect collagen fibrils from cleavage by collagenases.