Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli

Xing, Lei; Xu, Wen-Tao; Zhou, Bihong; Chen, Yilu; Lin, Zhanglin

doi:10.1016/j.pep.2013.01.012

Cited by 18 publications

(14 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For histatin 1, the yield was about 2‐6‐fold higher than that obtained with the traditional cSAT method before RP‐HPLC purification, and about 1.2–3.3‐fold higher after purification. These yields following RP‐HPLC purification also compared favorably with those obtained using the GST‐tag purification strategy (2.4–6.7 mg/L vs. 4.0 mg/L) . For GLP‐1, the yields were about 0.9–2.0‐fold higher than with the traditional cSAT method before RP‐HPLC purification, and about 0.5–1.9‐fold higher after purification.…”

Section: Discussionmentioning

confidence: 70%

“…A) . The yield was estimated to be 4.1 ± 0.1 μg/mg wet cell weight, which was about two‐fold higher than that obtained using the traditional cSAT method . The purity was estimated to be 90% with only a small amount of disaggregated L 6 KD‐I fragment being retained.…”

Section: Resultsmentioning

confidence: 84%

“…On SDS-PAGE analysis, the histatin 1 sample migrated slower than expected for a molecular mass of about 6.5 kD (Fig. 3A) [26]. The yield was estimated to be 4.1 ± 0.1 μg/mg wet cell weight, which was about twofold higher than that obtained using the traditional cSAT method [26].…”

Section: Production Of Histatin 1 and Glp-1 In The L 6 Kd-mentioning

confidence: 83%

See 2 more Smart Citations

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Zhao

Zhou

Gao

et al. 2017

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self-assembling peptides and a C-terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self-assembling peptide. Upon simple separation, the target protein or peptide with an authentic N-terminus is then released in the solution by intein-mediated cleavage. Different combinations of four self-assembling peptides (ELK16, L KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI-CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI-CM, which has pH-shift inducible cleavage, was found to work well with three self-assembling peptides (L KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx-strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N-terminus, which is especially effective for peptides of 30-100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.

show abstract

Section: Discussionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 84%

See 1 more Smart Citation

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Zhao

Zhou

Gao

et al. 2017

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…The yields are between 4.3 and 27 mg/L culture at the test tube level (Table 1) [10, 41, 43]. The method has also been used to express and purify the antimicrobial peptide Histatin 1, with a yield of 5.48 mg/L culture (Table 1) [44].…”

Section: Purification Tags That Induce Active Protein Aggregates Inmentioning

confidence: 99%

Aggregating tags for column‐free protein purification

Lin

Zhao

Xing

et al. 2015

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

Protein purification remains a central need for biotechnology. In recent years, a class of aggregating tags has emerged, which offers a quick, cost-effective and column-free alternative for producing recombinant proteins (and also peptides) with yield and purity comparable to that of the popular His-tag. These column-free tags induce the formation of aggregates (during or after expression) when fused to a target protein or peptide, and upon separation from soluble impurities, the target protein or peptide is subsequently released via a cleavage site. In this review, we categorize these tags as follows: (i) tags that induce inactive protein aggregates in vivo; (ii) tags that induce active protein aggregates in vivo; and (iii) tags that induce soluble expression in vivo, but aggregates in vitro. The respective advantages and disadvantages of these tags are discussed, and compared to the three conventional tags (His-tag, maltose-binding protein [MBP] tag, and intein-mediated purification with a chitin-binding tag [IMPACT-CN]). While this new class of aggregating tags is promising, more systematic tests are required to further the use. It is conceivable, however, that the combination of these tags and the more traditional columns may significantly reduce the costs for resins and columns, particularly for the industrial scale.

show abstract

“…Truncated RNAse, truncated Hpal Paramoysin, Thioredoxin Trx RNAP CM4, HβD4 Hstl Anophelin CHAP Pa‐MAP 2 Thymosin B4…”

Section: Introductionmentioning

confidence: 99%

Inteins as tools for tagless and traceless protein purification

Lahiry

Fan

Stimple

et al. 2017

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

The purification of recombinant proteins is a complicated process that requires a thorough understanding of the physical and chemical properties of each protein of interest. The unique characteristics of each protein require the development of a complicated, multi-step process consisting of several orthogonal chromatographic techniques. Although affinity tag methods have been useful in simplifying this process, these approaches have significant drawbacks when tagless proteins are required. Therefore, the development of a flexible, economical, and efficient purification platform for traceless and tagless target proteins would represent a significant advance in bioprocess development. Self-cleaving tags have enabled purification of a broad range of target proteins using simple affinity approaches, but with the ability to ultimately deliver a tagless target protein. Thus these tags potentially offer a purification platform analogous to Protein A, but without the limitation to antibody targets. This review summarizes the advances in developing various intein-based self-cleaving tag technologies, their preferred cleavage conditions (reducing agents, pH, temp, etc.) and the effect of different target proteins on intein catalytic activity. We also discuss engineered inteins whose activity (protein splicing or cleavage) is stringently controlled/triggered by small molecules, light, or environmental condition such as salt concentration.

show abstract

Facile expression and purification of the antimicrobial peptide histatin 1 with a cleavable self-aggregating tag (cSAT) in Escherichia coli

Cited by 18 publications

References 45 publications

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Aggregating tags for column‐free protein purification

Inteins as tools for tagless and traceless protein purification

Contact Info

Product

Resources

About