Facile expression and purification of active human growth hormone in E. coli by a cleavable self-aggregating tag scheme

Lin, Zhanglin; Ndengue, Peguy Paulie Amesso; Jing, Yanyun; Zhao, Lei; Yang, Xiaofeng

doi:10.1016/j.pep.2021.105974

Cited by 9 publications

(10 citation statements)

References 41 publications

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“…The precipitated fusion protein was then resuspended in a solution at pH 6.2 (containing 0.7 M Na 2 SO 4 ) to induce C-terminal cleavage of Mtu ΔI-CM, releasing Di-GLP-1 or Tri-GLP-1 into the supernatant. We further purified Di-GLP-1 or Tri-GLP-1 using a two-step standard column purification, i.e ., IEC (ion exchange chromatogram, anion exchange resin) and SEC (size exclusion chromatogram, Superdex 75 resin). , …”

Section: Resultsmentioning

confidence: 99%

“…We further purified Di-GLP-1 or Tri-GLP-1 using a two-step standard column purification, i.e., IEC (ion exchange chromatogram, anion exchange resin) and SEC (size exclusion chromatogram, Superdex 75 resin). 29,30 As shown in Figure 1C,D, the yields for icSAT-tagged GLP-1-SpyTag, Di-SpyCatcher(ΔN), and Tri-SpyCatcher(ΔN) were 203, 517, and 427 mg/L LB at the shake-flask scale, respectively. We observed that 67% of the icSAT-tagged GLP-1-SpyTag was prematurely cleaved, even when using the Mtu ΔI-CM variant (Figure 1C,D, line ES1).…”

Section: Design and Preparation Of Di-glp-1 Or Tri-glp-1mentioning

confidence: 92%

“…All three proteins were further purified by a two-step chromatography as previously described using an ÄKTA pure protein purification system (GE Healthcare, USA) with minor modifications. , The resins and pH used in the IEC and SEC steps are given in Table S1. Briefly, the supernatant fractions containing Di-GLP-1, Tri-GLP-1, or GLP-1/GIP were pooled and dialyzed against buffer B5 (20 mM Tris-HCl, pH 7.2) and then loaded onto a UniGel-30Q anion exchange column (Suzhou NanoMicro Technology Co., Ltd., Jiangsu, China) and eluted with a 5-column volumes (CV) linear gradient of 0–0.2 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

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Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

Zheng,

Lao,

Liu

et al. 2024

Bioconjugate Chem.

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The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable selfaggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher-and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0-to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8-to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.

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Section: Resultsmentioning

confidence: 99%

Section: Design and Preparation Of Di-glp-1 Or Tri-glp-1mentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

Zheng,

Lao,

Liu

et al. 2024

Bioconjugate Chem.

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“…hGH produced by Zhanglin Lin et al combined with L6KD maintained 92% bioactivity. This study provides a simple and economical method to efficiently produce active rhGH [8] (Figure 1). ELK16 was further studied by JufangWang et al They compared three different methods of producing CeA peptide: a Histag-fused protein expression system (AT-HIS system), a cell-free protein expression system (CF system), and a selfassembling peptide-fused protein expression system (SA-ELK16 system).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Applications of self-aggregating peptide as a novel bio-nanomaterial

Zhu¹

2023

Second International Conference on Biological Engineering and Medical Science (ICBioMed 2022)

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Peptides are widely found in natural organisms and play a variety of biological functions in human body. Compared with traditional materials, peptides have lower immunogenicity and better biocompatibility, which makes them ideal biological materials. Self-aggregating peptide is a kind of peptide chain that can be assembled by covalent binding of monomers under physiological or pathological conditions. Their advantages include the ability to aggregate by themselves and their low immunogenicity and biocompatibility as peptides. Therefore, self-aggregating peptides have various potential applications in biology and medical field. Since self-aggregating peptide can self-assemble into insoluble particles under certain conditions, wrapping the target protein with self-aggregating peptide can achieve the purpose of protein purification by simple centrifugation. This method is superior to the existing protein purification methods, since the experimental efficiency and cost can be optimized. Moreover, self-aggregating peptides, because of their peptide nature, do not elicit strong immune responses. Thus, self-aggregating peptides can be loaded with a large number of drugs and can be biodegraded into unharmful products which can be smoothly absorbed by the human bodies. The self-aggregated peptide loaded subunit vaccine can successfully induce and enhance immune response without adverse reactions, which is superior to existing immune adjuvants. Self-aggregated peptide also helps keeping the vaccine active for a long time and effectively reduce the cost of vaccine promotion. Select self-aggregating peptides that can be cleaved under pathological conditions to load drugs can help achieve drug targeting. In addition, the self-aggregating peptides loaded with drugs can be stored in specific sites for a long time, so that it does not require repeated dosing, thus improving the efficiency of drug administration. In this paper, the design of peptide self-assembly based solutions and their different biomedical applications are summarized, and their future prospects are prospected.

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“…AIBs were used with high efficiency to produce several proteins and peptides including human histatin 1 (Xing et al, 2013; Zhao et al, 2017), human glucagon‐like peptide 1 (Zhao et al, 2016; Zhao et al, 2017), growth hormone (Lin et al, 2021), as well as l ‐lysine decarboxylase from E. coli (Kloss et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of biologically active proteins as L6KD‐SUMO fusions forming inclusion bodies in Escherichia coli

Komolov,

Sannikova,

Gorbunov

et al. 2023

Biotech & Bioengineering

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A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N‐terminal self‐associating L6KD peptide fused to the SUMO protein (small ubiquitin‐like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6KD‐SUMO fusions, and the resulting three‐component fusion proteins are synthesized and self‐assembled in E. coli into so‐called active inclusion bodies (AIBs). In vivo, the L6KD‐SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO‐specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling “loofah sponges” with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

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Facile expression and purification of active human growth hormone in E. coli by a cleavable self-aggregating tag scheme

Cited by 9 publications

References 41 publications

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

Customizable Click Biochemistry Strategy for the Design and Preparation of Glucagon-like Peptide-1 Conjugates and Coagonists

Applications of self-aggregating peptide as a novel bio-nanomaterial

Synthesis of biologically active proteins as L6KD‐SUMO fusions forming inclusion bodies in Escherichia coli

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