Self-encapsulation and controlled release of recombinant proteins using novel silica-forming peptides as fusion linkers

Abdel‐Hamid, Mohamed; Yeo, Ki Baek; Ki, Mi-Ran; Pack, Seung Pil

doi:10.1016/j.ijbiomac.2018.12.160

Cited by 33 publications

(14 citation statements)

References 45 publications

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“… 28 Here, we demonstrate the encapsulation of the active integral membrane protein DGK in biomimetic silica with the aim to extend the use of functionally encapsulated IMPs to applications as catalysts in biotechnology or as sensors in biomedical applications, similar to the scope of applications for encapsulated soluble enzymes. 29 , 30 …”

Section: Introductionmentioning

confidence: 99%

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis . Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic β-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

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Section: Introductionmentioning

confidence: 99%

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Bialas

Becker

2021

Bioconjugate Chem.

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“…For elution l ‐lysine was chosen because, as previously stated, NaCl is not suitable to achieve good elution and amino acids such as lysine and arginine showed a preferable result [19, 28, 29, 38]. Using these amino acids has the additional advantage of stabilizing eluted proteins [39, 40].…”

Section: Resultsmentioning

confidence: 99%

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Rauwolf

Steegmüller

Schwaminger

et al. 2021

Engineering in Life Sciences

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Silica is widely used for chromatography resins due to its high mechanical strength, column efficiency, easy manufacturing (i.e. controlled size and porosity), and low‐cost. Despite these positive attributes to silica, it is currently used as a backbone for chromatographic resins in biotechnological downstream processing. The aim of this study is to show how the octapeptide (RH)4 can be used as peptide tag for high‐purity protein purification on bare silica. The tag possesses a high affinity to deprotonated silanol groups because the tag's arginine groups interact with the surface via an ion pairing mechanism. A chromatographic workflow to purify GFP fused with (RH)4 could be implemented. Purities were determined by SDS‐PAGE and RP‐HPLC. The equilibrium binding capacity of the fusion protein GFP‐(RH)4 on silica is 450 mg/g and the dynamic binding capacity around 3 mg/mL. One‐step purification from clarified lysate achieved a purity of 93% and a recovery of 94%. Overloading the column enhances the purity to >95%. Static experiments with different buffers showed variability of the method making the system independent from buffer choice. Our designed peptide tag allows bare silica to be utilized in preparative chromatography for downstream bioprocessing; thus, providing a cost saving factor regarding expensive surface functionalization. Underivatized silica in combination with our (RH)4 peptide tag allows the purification of proteins, in all scales, without relying on complex resins.

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“…Generally, a protein with a pI of >7.0 often had the ability of biosilicification. , For the proteins unable to mediate silica formation, one could confer them with the ability of silica precipitation through the SMPs fusion using recombinant DNA technology. ,,, BglS had a pI value of 5.77. Accordingly, it was reasonable to believe that BglS needed the help of a SMP for silicification-based self-immobilization.…”

Section: Resultsmentioning

confidence: 99%

Investigation of the ELP-Mediated Silicification-Based Protein Self-Immobilization Using an Acidic Target Enzyme

Lin

Qiu

Cai

et al. 2020

Ind. Eng. Chem. Res.

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Recently, we proposed a novel and effective strategy for enzyme immobilization, namely, as elastin-like polypeptide (ELP)-mediated silicification-based protein self-immobilization (ESPSI). Here, an enzyme with an acidic isoelectric point (pI) was chosen to further demonstrate the feasibility and generality of this method; pI is a critical factor in biosilicification. The lichenase (BglS) from Bacillus subtilis 168, with a theoretical pI of 5.77, was genetically fused to the cationic ELP. The recombinant chimera protein BglS-ELP (B-E) was purified through ELP-based nonchromatographic inverse transition cycling (ITC) method. B-E then was self-encapsulated within silica nanoparticle (NP) via ELP-mediated biomimetic silicification, producing the nanobiocatalyst B-E@silica. Excellent encapsulation efficiency (>85%) could be achieved within a short immobilization time (10 min). In addition, the encapsulation efficiency could be promoted through temperature-induced phase transition. Meanwhile, this method gave negligible protein leakage (<0.5%). Thus, the B-E@silica showed good reusability, retaining ∼80% of the initial activity after 10 reaction cycles. These results indicated that ESPSI was suitable for the protein with an acidic pI. In addition, phase transition was a unique route to improve immobilization efficiency.

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Self-encapsulation and controlled release of recombinant proteins using novel silica-forming peptides as fusion linkers

Cited by 33 publications

References 45 publications

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Biomimetic Silica Encapsulation of Lipid Nanodiscs and β-Sheet-Stabilized Diacylglycerol Kinase

Purification of a peptide tagged protein via an affinity chromatographic process with underivatized silica

Investigation of the ELP-Mediated Silicification-Based Protein Self-Immobilization Using an Acidic Target Enzyme

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