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Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G ColG and H ColH from Clostridium histolyticum and collagenase T ColT from Clostridium tetani , mimicking the isoforms in vivo.
Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant. This production and purification platform paves the way for systematic screenings of recombinant collagenases to enlighten the biochemical function and to identify key residues and motifs in collagenolysis.
Clostridia comprise a diverse family of anaerobic, sporulating bacteria, including notorious pathogenic species such as Clostridium botulinum , Clostridium perfringens , and Clostridium difficile.
Two further prominent representatives are Clostridium histolyticum , a pathogen-causing gas gangrene, and Clostridium tetani giving rise to tetanus Bruggemann et al. While the histotoxicity of Clostridia is primarily caused by specific toxins Hatheway , host infiltration and colonization are triggered by the production of various proteases such as collagen degrading zinc-metalloproteases, namely collagenases Bruggemann and Gottschalk ; Hatheway ; Mallya et al.
Therefore, clostridial collagenases have been proposed as important drug-target candidates. Additionally, with collagens being the most abundant proteins in all higher organisms, there exists a diverse spectrum of therapeutic and biotechnological applications for bacterial collagenases, in particular for the biochemically well-characterized ColG and H from C. They are mosaic proteins consisting of a signal peptide, a catalytic domain, up to two polycystic kidney disease domain s PKD of unknown function and up to three collagen binding domain s CBD; see Fig.
Their domain organization varies significantly. Schematic representation of the domain architecture of three different mature clostridial collagenases. Contrasting the zinc coordination in metzincins e. The structural knowledge about the clostridial collagenases is poor and currently confined to the crystal structure of the CBD Wilson et al. The high biotechnological and medical interest in clostridial collagenases that is mirrored by an increasing demand for high grade mono-isoformic preparations, in particular of ColG and ColH, faces a shortage of supply due to 1 the pathogenic origin of ColH and ColG and 2 the intricacy associated with the purification of homogeneous isoforms.
The development of simple and efficient high-level expression and purification strategies would also facilitate basic and applied research of bacterial collagenases. Reported recombinant expression trials focused on Bacillus subtilis , C.
Although the latter expression system was considered as inefficient in translating clostridial genes Tanaka et al. The established protein expression and purification platform provides cost-efficient access to these biotechnologically important enzymes and paves the way for systematic enzyme engineering approaches. In our study, we selected three bacterial collagenases: ColG, H, and T that are characterized by complementary domain architectures Fig.
Schematic representation of the expression vectors. Lowercase letter indicates the cleavable N-terminal tag h: His6, m: MBP , capital letter the respective collagenase G, H or T , and the digit denotes the number of domains present in the construct.
Temp expression temperature; POE period of expression. Restriction enzymes and T4 Ligase were obtained from Fermentas, St. Leon-Rot Germany. All reagents were of the highest standard available from Sigma—Aldrich Co. All inserts were cloned in a modified pET15b expression vector encoding for an N-terminal His 6 -tag followed by a TEV Tobacco Etch Virus protease cleavage site for specific tag removal.
All constructs were confirmed by DNA sequencing prior to protein expression. Plasmids were introduced into expression hosts via electroporation. Parameters considered for optimization in terms of maximizing soluble expression were: 1 different E. In sum, 64 different expression conditions were tested for every construct. Parameters found optimal were used for large-scale expressions. Large-scale expression was carried out in the respective E.
Purification via the MBP tag was carried out in batch mode. Application of the sample occurred at a flow rate of 0. A prestained protein ladder was used as size marker Fermentas , and gels were stained with Coomassie Brilliant Blue R To gain insights into the structure—function relationship of the clostridial collagenases G, H, and T, the proteins were dissected by constructing different recombinant derivatives of the full length enzymes.
To delineate the appropriate boundaries of the different domains, we combined the following data: 1 information on the mature N-terminus as described in the literature Matsushita and Okabe , 2 information on naturally occurring isoforms, as described for ColG and ColH from C. Thereby, the initial construct design mimics the domain organization of collagenase isoforms in vivo. Due to observed protein degradation during protein purification data not shown seven constructs, three of ColG, and ColH each and one of ColT were subcloned into the pMBP-Parallel2 vector Sheffield et al.
To maximize soluble protein expression, 64 different expression conditions, based on five different parameters host strain, point of induction, final IPTG concentration, period, and temperature of expression were screened for every construct. A representative expression gel of all constructs cloned in pETb is shown in Fig.
M prestained protein ladder. To achieve pure and homogeneous protein samples, it was necessary to combine diverse purification methods Fig. We, therefore, established a flexible purification platform adaptable to the individual needs of the investigated constructs. We utilized not only the advantages of well-characterized fusion tags His 6 - and MBP-tag but also capitalized on intrinsic properties of the proteins, e.
As a final polishing step SEC was routinely used. Schematic representation of the modular purification platform. But in most cases, at least one additional orthogonal purification step via IEC had to be applied Fig.
For the full length constructs, even a tandem affinity approach combined with IEC and SEC was necessary to successfully bypass co-purification of C-terminally degraded variants Fig. N-terminal fusion tags were routinely removed with TEV protease before or after the ion exchange chromatography step. Differential purification strategy exemplified by hG1, hG2, and mG4. To summarize the individual purification steps: 1 because all constructs featured an N- or C-terminal His 6 -tag, immobilized metal affinity chromatography IMAC was by default used as initial purification step Fig.
Most E. For this purpose we established a calcium gradient ion exchange chromatography, which capitalizes on the specific calcium binding properties of these enzymes. All proteins migrated as monomers at the expected molecular size.
A representative SEC run is shown in Fig. Purification of the catalytic domain of ColT hT1. Lanes 1 , 2 flow-through containing the target protein without His 6 -tag; lanes 3 , 4 wash steps; lane 5 elutions containing the TEV-protease; d Size exclusion chromatography of the catalytic domain without His 6 -tag.
All variants except the two mini-catalytic domains were active against the synthetic substrate. As expected, enzyme activity of all variants could be reversibly inhibited by the zinc-specific inhibitor 1,phenanthroline data not shown. The continuing progress in biotechnological and medical research is uncovering an ever-growing number of possible applications for clostridial collagenases, in particular ColG and H Antonioli et al.
There are various commercial preparations available, but these usually contain several collagenase isoforms and even other proteases e. Their heterogeneity and the difficulty of obtaining mono-isoformic preparations have so far hindered us from exhausting their full biotechnological and medical potential, such as the development of application-specific mutants and rational drug design.
We employed the working horse of protein expression— E. To optimize the yield of soluble protein, we tested several expression parameters for all constructs: 1 E. All monitored parameters affected the soluble protein yield, justifying this comprehensive screening of heterologous expression as the first point of optimization in protein production and purification. In a second step, we optimized the purification protocols based on a flexible arrangement of different chromatographic techniques affinity, ion exchange and size exclusion chromatography to the individual needs of the investigated isoforms Fig.
For instance, a two step purification approach was already sufficient for shorter constructs, e. The revealed auto degradation pattern of the full-length constructs apparently correlated roughly with the size of the naturally occurring isoforms and, therefore, gave additional impetus to the idea to mimic their mosaic domain architecture with our constructs.
This approach enabled us to pool nondegraded protein in the successive purification steps. In response to the presence of a truncated fragment smaller than the catalytic domain construct in preparations of ColT, two shorter variants were cloned to identify an even smaller active catalytic domain. However, both mini-constructs showed no catalytic activity in the FALGPA assay, although they migrated on SEC at the expected size, indicative of proper folding data not shown.
Therefore, we consider the present S1 variants as minimal versions of the catalytic domains. These observations, 1 C-terminally truncated isoforms in vivo Bond and Van Wart a ; Matsushita et al. In conclusion, we succeeded in establishing a generic expression and purification strategy for clostridial collagenases in E. This system evades the problems associated with homologous expression i.
Based on the old but by far not outdated workhorse E. Moreover, based on this platform, a systematic screening of chimeric constructs and of other mutants is feasible which will help to identify key residues and motifs in collagenolysis and enlighten the biochemical function of the individual domains present in clostridial collagenases.
Hans Brandstetter, Email: ta. National Center for Biotechnology Information , U. Applied Microbiology and Biotechnology. Appl Microbiol Biotechnol. Published online Mar Author information Article notes Copyright and License information Disclaimer. Corresponding author.
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This article has been cited by other articles in PMC. Abstract Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine.
Keywords: Clostridial collagenases, Expression, Purification, Platform. Introduction Clostridia comprise a diverse family of anaerobic, sporulating bacteria, including notorious pathogenic species such as Clostridium botulinum , Clostridium perfringens , and Clostridium difficile. Open in a separate window. Expression of ColG, H, and T Test expression and solubility testing Plasmids were introduced into expression hosts via electroporation.
Large-scale expression and cell harvest Large-scale expression was carried out in the respective E. Amylose affinity chromatography Purification via the MBP tag was carried out in batch mode. Results Construct design and cloning To gain insights into the structure—function relationship of the clostridial collagenases G, H, and T, the proteins were dissected by constructing different recombinant derivatives of the full length enzymes.
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Clostridial collagenases are foe and friend: on the one hand, these enzymes enable host infiltration and colonization by pathogenic clostridia, and on the other hand, they are valuable biotechnological tools due to their capacity to degrade various types of collagen and gelatine. However, the demand for high-grade preparations exceeds supply due to their pathogenic origin and the intricate purification of homogeneous isoforms. We present the establishment of an Escherichia coli expression system for a variety of constructs of collagenase G ColG and H ColH from Clostridium histolyticum and collagenase T ColT from Clostridium tetani , mimicking the isoforms in vivo. Based on a setup of five different expression strains and two expression vectors, 12 different constructs were expressed, and a flexible purification platform was established, consisting of various orthogonal chromatography steps adaptable to the individual needs of the respective variant.
William Astbury described molecular biology in in Nature , as:. It is concerned particularly with the forms of biological molecules and [ It must at the same time inquire into genesis and function.
Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. There is no doubt that the production of recombinant proteins in microbial systems has revolutionized biochemistry.
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