Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins

Miraula, Manfredi; Enculescu, Charmaine; Schenk, Gerhard; Mitić, Nataša

doi:10.4236/ajmb.2015.52005

Cited by 10 publications

(5 citation statements)

References 106 publications

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“…Although long considered nothing more than the products of parasitic genetic elements (3), recent work has shown that some inteins function as environmental sensors to modulate host protein function in response to environmental changes (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Identifying cellular or environmental factors that influence intein splicing, and thus regulation of essential protein functions, is an emerging area of interest in both basic and applied research fields (16)(17)(18).…”

mentioning

confidence: 99%

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Woods

Vangaveti

Egbanum

et al. 2020

mBio

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Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis. We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis. Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae. Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc. IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

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mentioning

confidence: 99%

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Woods

Vangaveti

Egbanum

et al. 2020

mBio

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“…The amino acid residue at position +1 consists either of a cysteine, serine, or threonine, depending on the split intein. PTS takes place in four steps: (1) a nucleophilic attack by the first amino acid residue of either a cysteine or serine located in the N-terminal intein part on the carbonyl carbon of the preceding amino acid residue (−1 position) located in the flanking N-extein leads to an N-S or N-O acyl shift; (2) a linear (thio) ester intermediate being trans-esterificated by a nucleophilic attack of the first amino acid residue of the so-called C-extein (+1 position) forms a branched intermediate [95]; (3) the N-terminal intein part is now cleaved from its fusion protein and transferred to the N-extein. As a result, a succinimide ring is generated through cyclization of the conserved asparagine residue of the C-terminal intein part after the nucleophilic attack of the previously formed intein extein junction; (4) finally, the cleavage reaction of the C-terminal intein part followed by a spontaneous S-N or O-N acyl shift ligates the esterified C-and N-exteins by a native stable peptide bond [96,97].…”

Section: Split Inteinsmentioning

confidence: 99%

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Hofmann

Krah

Sellmann

et al. 2020

IJMS

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Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

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“…İnteinlerin kendiliğinden proteinden ayrılma özelliğinin belirlenmesi ile birlikte inteinler protein saflaştırmasında kullanılmaya başlanmıştır. İnteinler herhangi bir polipeptitten kimyasal olarak ayrılabilme veya peptitleri birbirine bağlayabilme özelliklerinden dolayı birçok moleküler çalışmada kullanılmaktadırlar [8][9][10][11]. Doğal halde organizmalarda bulunan inteinler ilk defa Saccharomyces cerevisiae VMA1 geninden elde edilmiştir [12,13].…”

Section: İntei̇nunclassified

A Review on Intein Mediated Recombinant Protein Purification Processes Without Use of Protease

Ermurat

2017

ANADOLU UNIVERSITY JOURNAL OF SCIENCE AND TECHNOLOGY –C Life Sciences and Biotechnology

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Bu derleme çalışmasında, yeniden birleştirilmiş proteinlerin proteaz kullanılmadan saflaştırılması süreçlerinde yararlanılan inteinlerin ayrılma etkinliklerinin artırılması için inteinler ve hedef proteinler üzerinde yapılmış araştırmalar incelenmiştir. İntein kullanımı ile protein ayrışması üzerinde yapılan araştırmalar, intein ve protein saflaştırma süreçlerinin daha verimli hale getirilmesini amaçlamaktadır. In vivo ve in vitro olarak gerçekleşen süreçlerde, intein ve hedef protein içeren poliklonal Polimeraz Zincir Tepkimesi (PZT) ürününün çoğaltılması ve ekspres plazmidlerin kopyalanan suşlara taşınması işlemleri yapılmaktadır. İnteinin proteinden ayrılma etkinliği ve ayrılma oranı sabiti kinetikleri, değişik pH, sıcaklık ve süzme sürelerinde belirlenmekte ve hedef proteinlere ilişkili olarak karşılaştırılmaları yapılmaktadır. Araştırmalarda değişik doğal ve yapay inteinlerin protein ayrıştırma etkinlikleri incelenmiş ve yapay Nostoc punctiforme (Npu) DnaE C-inteinlerinin ayrışma etkinliklerinin en yüksek olduğu belirlenmiştir. Çalışmalar amino asit eklenmesine daha yatkın olması nedeniyle Cinteinler üzerinde yoğunlaşmaktadır.

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Inteins—A Focus on the Biotechnological Applications of Splicing-Promoting Proteins

Cited by 10 publications

References 106 publications

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

A Review on Intein Mediated Recombinant Protein Purification Processes Without Use of Protease

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