Fusion tags to enhance heterologous protein expression

Ki, Mi-Ran; Pack, Seung Pil

doi:10.1007/s00253-020-10402-8

Cited by 126 publications

(93 citation statements)

References 140 publications

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“…Heterologous expression protocols with solubility tags are widely used in the literature [52][53][54][55]. These different tags help in the correct folding of protein, solubilization, purification, and protecting the recombinant peptides against the degradation produced by intracellular proteases [56]. MBP tag is a frequently used fusion tag to enhance molecule solubility [57] and is also capable to interact with hydrophobic amino acid residues present in unfolded proteins [58].…”

Section: Discussionmentioning

confidence: 99%

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Ramos¹,

Rangel²,

Andrade³

et al. 2021

J. Venom. Anim. Toxins incl. Trop. Dis

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Background Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive ( Bacillus thuringiensis ) and gram-negative ( Burkholderia kururiensis and E. coli ) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro .

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Section: Discussionmentioning

confidence: 99%

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Ramos¹,

Rangel²,

Andrade³

et al. 2021

J. Venom. Anim. Toxins incl. Trop. Dis

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“…Several peptide tags, such as the FLAG (Xiao et al, 2019;Cui et al, 2020;Miura et al, 2020;Uemura et al, 2020), HA (Shinozawa et al, 2019;Kuhnert et al, 2020), and c-MYC (Earley et al, 2006;Baudisch et al, 2018;Roshan et al, 2018) tags are used in plants because of their specificity. These tags may affect the solubility or insolubility of the expressed proteins and their activities, depending on the characteristics of the expressed proteins (Ki and Pack, 2020). Thus, availability of different protein tagging systems is desirable.…”

Section: Discussionmentioning

confidence: 99%

RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

et al. 2020

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An affinity tag system requires both high affinity and specificity. The RAP tag epitope DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is specifically recognized by PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2 possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a useful tagging system for detecting and purifying proteins from plant cells. The heavy chain of PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the light chain of the antibody were introduced into N. benthamiana by agroinfiltration. The expression of PMab-2 peaked 4 days after agroinfiltration, and approximately 0.3 mg/g fresh weight of the antibody was accumulated. After purification, the plant-derived PMab-2 successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost the same binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed in plant cells were specifically recognized by PMab-2. These results indicate that PMab-2 can accumulate at high levels in N. benthamiana and is easily purified and that the RAP tagging system presents a useful tool for detecting and purifying proteins of interest in plant cells.

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“…As another strategy, solubility enhancers, such as maltose-binding protein (MBP) [1,17], oleosin [12], low-molecular-weight protamine [14], HaloTag [18], glutathione S-transferase [1,17], protein disulfide isomerase (PDI) [1,17], ELK16 [29], and thioredoxin [1,17,30], are fused to the target protein, including GFs. As the solubility of the tag affects the solubility of the passenger protein, choosing an appropriate solubility tag is critical [31]. Nguyen et al [1,17] reported that seven protein tags, namely, thioredoxin, glutathione S-transferase, N-utilization substance protein A, the b'a'domain of PDI (PDIb'a'), PDI, 6×His, and MBP, were tested, and some or all of the protein tags enhance the solubility of the GFs in E. coli.…”

Section: ]mentioning

confidence: 99%

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

et al. 2021

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Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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Fusion tags to enhance heterologous protein expression

Cited by 126 publications

References 140 publications

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

RAP Tag and PMab-2 Antibody: A Tagging System for Detecting and Purifying Proteins in Plant Cells

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

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