Site-Specific and High-Loading Immobilization of Proteins by Using

Jun 29, 2016 - Immobilization of enzymes enhances their properties for application in industrial processes as reusable and robust biocatalysts. Here, ...
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Site-Specific and High-Loading Immobilization of Proteins by Using Cohesin−Dockerin and CBM−Cellulose Interactions Mei Li, Yi Yue, Zi-Jian Zhang, Zai-Yu Wang, Tian-Wei Tan, and Li-Hai Fan* Beijing Key Laboratory of Bioprocess. College of Life Science and Technology, Beijing University of Chemical Technology, Beisanhuan East Road #15, Beijing, China 100029 S Supporting Information *

ABSTRACT: Immobilization of enzymes enhances their properties for application in industrial processes as reusable and robust biocatalysts. Here, we developed a new immobilization method by mimicking the natural cellulosome system. A group of cohesin and carbohydrate-binding module (CBM)-containing scaffoldins were genetically engineered, and their length was controlled by cohesin number. To use green fluorescent protein (GFP) as an immobilization model, its Cterminus was fused with a dockerin domain. GFP was able to specifically bind to scaffoldin via cohesin−dockerin interaction, while the scaffoldin could attach to cellulose by CBM− cellulose interaction. Our results showed that this mild and convenient approach was able to achieve site-specific immobilization, and the maximum GFP loading capacity reached ∼0.508 μmol/g cellulose.

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degradation of plant cell wall polysaccharides.22−25 The definitive characteristic of the cellulosome is the presence of a large cohesin-containing subunit (called scaffoldin), which is devoid of enzymatic activity and to which the dockerincontaining catalytic subunits are attached. The cohesin− dockerin interaction is species-specific,26 and it mediates cellulosome assembly with an affinity constant well above 109 M−1,27,28 which is one of the strongest protein−protein interactions that have been documented. Most scaffoldins contain between six and nine cohesins; thus, they can easily multiply the number of catalytic subunits that are incorporated in the cellulosome. In general, scaffoldins also contain a noncatalytic carbohydrate-binding module (CBM). The function of CBM is to specifically recognize and tightly bind polysaccharides, bringing the cellulosome into close and prolonged vicinity with its substrate. The aim of this work was to develop a site-specific and highloading protein immobilization approach based on the strong cohesin−dockerin and CBM−cellulose interactions (see Figure 1). The green fluorescent protein (GFP) was selected as an immobilization model, while microcrystalline cellulose, a stable, safe, inert, and naturally occurring substance, was used as the solid support. As illustrated in Figure 1, only if a dockerin domain is present, the recombinant GFP can specifically recognize and attach to the cohesin domains on scaffoldin, thus achieving the

nzymes are biologically active proteins with fascinating prospects for application in chemical industries due to their high activity under mild conditions, and high selectivity and specificity.1,2 Immobilization of enzymes is a requisite in most instances for their use as biocatalysts, since immobilization permits the simple reuse of the protein and simplifies the overall design and performance control of the bioreactor.3−5 When properly designed, the immobilization of enzymes has also been a powerful tool to improve enzyme stability, and in certain cases even their activity or selectivity.6−8 Although hundreds of immobilization approaches have been reported,9−14 the efforts on design of new techniques are still growing, of which robust immobilization of proteins with sitespecific orientation and high loading capacity are two of the most important areas of interest.3,5,7,8,14−18 For solid based immobilization, multipoint covalent attachment is likely the most effective strategy,13,14 but it is difficult to allow immobilization of enzymes at a well-defined position, since the proteins are usually attached to the solid surface by uncontrolled chemical bonds.7 This random immobilization tends to cause uncontrolled conformational changes, which may lead to a significant loss of enzyme activity, and the disordered enzyme orientation may also reduce the accessibility of substrate to functional sites.19,20 On the other hand, low enzyme loading capacity is probably another problem in covalent immobilization, and one of the current solutions is to use the nanostructured materials, which have extremely large specific surface area, as supports.15−18 However, cost-effective synthesis of nanomaterials may be a grand challenge.21 In Nature, a cellulosome is a macromolecular machine that adheres to the cell surface of some bacteria for efficient © 2016 American Chemical Society

Received: June 2, 2016 Revised: June 26, 2016 Published: June 29, 2016 1579

DOI: 10.1021/acs.bioconjchem.6b00282 Bioconjugate Chem. 2016, 27, 1579−1583

Communication

Bioconjugate Chemistry

Theoretically, the binding efficiency of GFP-docCipA to the adjacent cohesin domains may decrease due to the steric hindrance, so the native flanking linker sequence (30 bp) of olpB was involved in the cloned olpB. In this work, we designed 6 different scaffoldins (I−VI). Each had an individual cohesin domain number, but all of them got an N-terminal CBM. The scaffoldins I−VI were overproduced in E. coli BL21 (DE3) with T7 expression system (pET22b) and IPTG. Then, they were purified as GFP-docCipA with HisTrap FF crude Column. According to the deduced amino acid sequences, the molecular weight of olpB is 19.6 kDa, while CBM is 25.4 kDa. Therefore, the expected molecular weights of scaffoldin I, II, III, IV, V, and VI were 45.0 kDa, 64.6 kDa, 84.2 kDa, 103.8 kDa, 123.4 kDa, and 143.0 kDa, respectively. As shown in Figure 3B, the SDSPAGE results indicate that soluble scaffoldin I−VI were formed and purified, but the molecular weights (∼45.0 kDa, ∼70.0 kDa, ∼100 kDa, ∼120 kDa, ∼140 kDa, ∼160 kDa) were generally larger than the predicted values, especially those with more than one cohesin domains. This discrepancy may be attributed to the highly nonglobular property of the longer scaffoldins.29 Initial efforts to verify the binding ability of scaffoldins to microcrystalline cellulose were carried out by using the crude scaffoldins I−VI (see Figure 4A). Our hypothesis was that the scaffoldins would be specifically adsorbed from the E. coli extract (soluble cytoplasmic fraction), since only they had CBM. After binding, the proteins on cellulose were analyzed (see Figure 4B). Interestingly, the SDS-PAGE results show that, with the exception of the proteins larger than scaffoldins, all others including scaffoldins were able to be coimmobilized on cellulose. Although the immobilization of scaffoldins seems much more efficient than those nonspecific proteins, we still cannot rule out the possibility that this difference in binding capacity may be caused by the difference in protein concentrations in the E. coli crude extract, since scaffoldin I− VI obviously had much higher expression level (see Figure 4A). To further prove that the attachment of scaffoldins to cellulose was mediated by the CBM, we fused a C-terminal DsRed tag to scaffoldin II or scaffoldin II* (no CBM), and the corresponding proteins were called scaffoldin II-DR and scaffoldinII*-DR, respectively (see Figure 2). DsRed (225 aa) is a 28 kDa brilliantly red fluorescent protein that was recently cloned from Discosoma coral,30 and in this work, it provided a visual aid for estimating the cellulose adsorption of scaffoldins.

Figure 1. Strategies for site-specific and high-loading GFP immobilization on microcrystalline cellulose. GFP was fused with a C-terminal dockerin domain, and it was attached to scaffoldin through cohesin−dockerin interaction. A CBM was fused to the N-terminus of scaffoldin; thus, the GFP−scaffoldin complex could be immobilized on the cellulose surface via CBM−cellulose interaction.

site-specific immobilization. Here, the gene of dockerin (docCipA, 492 bp) was cloned from Clostridium thermocellum, a cellulosome-producting bacterium. The native GFP gene (717 bp) without its stop codon was then fused with docCipA in plasmid pYD1 (see Figure 2), and a short Gly-Ser (GS) liner (17 aa) was inserted between them in order to increase the solubility of the GFP-docCipA fusion. The recombinant protein was overexpressed in Escherichia coli BL21 (DE3) with plasmid pETDuet-1 (T7 expression system, see Figure 2) as the parent vector and IPTG as the inducer, and then purified from the cytoplasmic fraction of E. coli with HisTrap FF crude Column. After SDS-PAGE, the purified GFP-docCipA gave a clear band with a molecular weight around 48.0 kDa (see Figure 3A), which is in good agreement with the value (48.2 kDa) calculated from the deduced amino acid sequence, indicating that the soluble form of GFP-docCipA fusion was successfully produced. The scaffoldin plays an important role in high-loading immobilization, since its cohesin number directly determines the fixed level of the GFP. Also, the attachment of the GFP− scaffoldin complex to cellulose surface depends on the CBM in the scaffoldin terminus. Here, the genes of cohesin domain (olpB, 522 bp) and CBM (711 bp) were both cloned from C. thermocellum, and they were then fused in plasmid pET22b to construct the recombinant genes for scaffoldins (see Figure 2).

Figure 2. Structure maps of the recombinant plasmids for expression of GFP-docCipA and scaffoldins. T7p means T7 promoter, while T7t is T7 terminator. His (Histidine) tag is for protein purification by HisTrap FF crude column. 1580

DOI: 10.1021/acs.bioconjchem.6b00282 Bioconjugate Chem. 2016, 27, 1579−1583

Communication

Bioconjugate Chemistry

Figure 3. SDS-PAGE analysis of the purified GFP-docCipA and scaffoldin I−VI. (A) Marker (lane 1), GFP-docCipA (lanes 2 and 3). (B) Marker (lane 1), scaffoldins I−VI (lanes 2−7).

fluorescent, while the cellulose treated with only GFP-docCipA and without scaffoldins did not show obvious green fluorescence. These results indicate that GFP-docCipA could not directly bind to the cellulose surface, and the fixed GFPdocCipA only possibly attached to the immobilized scaffoldins, suggesting that our hypothesis to use a CBM and olpBcontaining scaffoldin for site-specific immobilization of proteins on cellulose was workable. Theoretically, the immobilized level of GFP-docCipA is mainly determined by the olpB number on scaffoldin. The scaffoldin that has more olpB domains is able to provide more binding sites for GFP-docCipA, resulting a higher GFP loading. In this work, scaffoldins I, II, III, IV, V, and VI had 1, 2, 3, 4, 5, and 6 olpB domains, respectively; thus, the adsorption capacity should be scaffoldin I < II < III < IV < V < VI. As the length of the scaffoldin increased, the gradually strengthened green fluorescence on cellulose (see Figure 5B) proves this hypothesis. To further determine the immobilization level of GFP on cellulose, we applied a fluorescence spectrometer to detect the green fluorescence intensity, and the results were illustrated in Figure 5C. It shows that the immobilized GFP reached ∼0.159, ∼0.290, ∼0.395, ∼0.457, ∼0.495, and ∼0.508 μmol/g cellulose when using scaffoldins I−VI, respectively. Compared with scaffoldin I, the GFP immobilized on cellulose by using scaffoldin VI was increased to ∼3.2-fold, of which the theoretical value should be 6-fold. This discrepancy has at least two possible causes. On one hand, the larger scaffoldin would take up a larger space on the cellulose surface, and thus the molecular number of scaffoldins I that could be immobilized on cellulose was more than that of scaffoldin VI. On the other hand, the attachment of GFP-docCipA to the immobilized scaffoldins started from the outer to the inner olpB; thus, the olpB domains close to the cellulose surface on scaffoldin VI would definitely have much lower binding efficiency. In conclusion, we have developed a new approach for enzyme immobilization based on protein−protein and protein−cellulose interactions. Compared with multipoint covalent attachment, although the robustness and stabilization may not be improved, this one point immobilization method is able to offer a number of advantages. (1) Site-specific orientation can help the immobilized enzymes to avoid the uncontrolled conformational changes and ensure the accessibility of substrate to functional sites. (2) This method is a one step process, and it can simply achieve a high protein loading capacity on supports. (3) The large mobility of the immobilized enzymes perhaps can reduce the diffusion limitation. (4) Cohesin−dockerin interaction has been proven to be speciesspecific,26 so it may be possible to immobilize multienzymes with a highly ordered structure by using more different cohesins

Figure 4. Functional immobilization of scaffoldins on cellulose. (A) SDS-PAGE analysis of the unpurified scaffoldins. Marker (lane 1), scaffoldins I−VI (lanes 2−7). (B) SDS-PAGE analysis of the adsorption of unpurified scaffoldins. Immobilized scaffoldins I−VI (lanes 8−13). (C) SDS-PAGE analysis of the adsorption of purified scaffoldin II-DR and II*-DR (no CBM). Marker (lane 1), scaffoldin IIDR immobilization (lane 2), purified scaffoldin II-DR (lane 3), scaffoldin II*-DR immobilization (lane 4), purified scaffoldin II*-DR (lane 5). (D) scaffoldin II-DR or II*-DR-treated cellulose under ultraviolet.

Scaffoldin II-DR and scaffoldinII*-DR were both expressed in E. coli BL21 (DE3) with T7 expression system (pET22b) and IPTG, and then purified with HisTrap FF crude column (see Figure 4C, Lines 3 and 5). The SDS-PAGE results show that scaffoldin II-DR and scaffoldinII*-DR were ∼110 kDa and ∼85 kDa, which were also larger than the values (92.6 kDa and 67.2 kDa) calculated from the amino acid sequences. Adsorption of the purified scaffoldin II-DR and scaffoldinII*-DR on microcrystalline cellulose was then tested. As shown in Figure 4C (Lines 2, 4) and Figure 4D, both results suggest that only scaffoldin II-DR had strong adsorbility, indicating that the specific attachment of scaffoldin to cellulose relied on CBM. In an effort to avoid the nonspecific adsorption, the nonspecific sites on microcrystalline cellulose were preblocked with bovine serum albumin (BSA), and the purified GFPdocCipA and scaffoldins I−VI were used in immobilization. After adsorption of scaffoldins on cellulose, GFP-docCipA was loaded, and the SDS-PAGE results in Figure 5A show that GFP-docCipA was successfully fixed on cellulose. The confocal laser scanning microscope was then applied for detection of the immobilized GFP. As shown in Figure 5B, the celluloses treated with both scaffoldins and GFP-docCipA were all brightly 1581

DOI: 10.1021/acs.bioconjchem.6b00282 Bioconjugate Chem. 2016, 27, 1579−1583

Communication

Bioconjugate Chemistry

Figure 5. Site-specific and high-loading immobilization of GFP via scaffoldins. GFP-docCipA and scaffoldins I−VI were prepurified. (A) SDS-PAGE analysis of the proteins on cellulose. Marker (lane 1), scaffoldins I−VI + GFP-docCipA (lanes 2−7). (B) Confocal laser scanning microscopy. (C) Amount of immobilized GFP on cellulose.

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and dockerins. (5) Cellulose is the most abundant natural biopolymer, and it not only can be shaped into nanosize particles,31 but also can be easily decorated onto other support materials.32 However, the possible interactions between the incompletely rigidified enzymes may be a problem in some cases,7 and the N-terminal dockerin domain may hinder enzyme expression and affect enzyme activity. Therefore, our further work will focus on introducing extra covalent linkage for rigidification, and using genetically engineered flexible arms for functional production of enzyme−dockerin fusion.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00282.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funding from the National High Technology Research and Development Program of China (863 Program, grant 2014AA020522), and the National Natural Science Foundation of China (grant 21376023). We also thank Dr Huimin Zhao for providing the plasmid of pRS424-HXT7p-GFP-HXT7t, and Dr Zhi-Hua Gan for help with confocal laser scanning microscopy. 1582

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DOI: 10.1021/acs.bioconjchem.6b00282 Bioconjugate Chem. 2016, 27, 1579−1583