Towards the elimination of chromatography in protein purification: Expressing proteins engineered to purify themselves

Mee, Courtney; Banki, Mahmoud Reza; Wood, David

doi:10.1016/j.cej.2007.04.021

Cited by 16 publications

(6 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although conventional affinity technologies have greatly simplified recombinant protein production, resins, and buffers are still too expensive. Hence, the tag removal adds another layer of complexity and expense to the recombinant protein production process ( Mee et al, 2008 ; Li, 2011 ).…”

Section: Soluble Protein Production In Escherichia Colimentioning

confidence: 99%

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

et al. 2014

View full text Add to dashboard Cite

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system.

show abstract

Section: Soluble Protein Production In Escherichia Colimentioning

confidence: 99%

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

et al. 2014

View full text Add to dashboard Cite

show abstract

“…Currently effective strategies have been developed to recover enzyme activity from aggregate particles [23,56]. Active IBs have shown unique advantages, such as easy separation, greater stability, and reusability, and offer robustness in applications [32]. Fusion of the N-terminal peptide GFIL8 to the Ulp1 results in increased production of active IBs with higher resistance to limited proteolysis and less leakage at different storage temperatures [18].…”

Section: Discussionmentioning

confidence: 99%

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

Han

Zeng

Zhang

et al. 2020

Journal of Industrial Microbiology and Biotechnology

View full text Add to dashboard Cite

The formation of inclusion bodies (IBs) without enzyme activity in bacterial research is generally undesirable. Researchers have attempted to recovery the enzyme activities of IBs, which are commonly known as active IBs. Tyrosine phenol-lyase (TPL) is an important enzyme that can convert pyruvate and phenol into 3,4-dihydroxyphenyl-l-alanine (L-DOPA) and IBs of TPL can commonly occur. To induce the correct folding and recover the enzyme activity of the IBs, peptides, such as ELK16, DKL6, L6KD, ELP10, ELP20, L6K2, EAK16, 18A, and GFIL16, were fused to the carboxyl terminus of TPL. The results showed that aggregate particles of TPL-DKL6, TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16 improved the enzyme activity by 40.9%, 50.7%, 48.9%, 86.6%, and 97.9%, respectively. The peptides TPL-DKL6, TPL-EAK16, TPL-18A, and TPL-GFIL16 displayed significantly improved thermostability compared with TPL. L-DOPA titer of TPL-ELP10, TPL-EAK16, TPL-18A, and TPL-GFIL16, with cells reaching 37.8 g/L, 53.8 g/L, 37.5 g/L, and 29.1 g/L, had an improvement of 111%, 201%, 109%, and 63%, respectively. A higher activity and L-DOPA titer of the TPL-EAK16 could be valuable for its industrial application to biosynthesize L-DOPA.

show abstract

“…In addition to these papers, two patent applications were filed, and this work was included in a chapter on industrial applications of inteins 16 . A review paper with an undergraduate first author was also published 18 . Notably, this research has generated quite a lot of attention, and has led to a commentary on the PHB system by George Georgiou in Protein Science 19 , as well as short blurbs on the ELP system in C&E News 20 , The Scientist 21 , Chemistry World 22 , and several online research discussion groups.…”

Section: Summary Of Most Important Resultsmentioning

confidence: 99%

Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Wood¹

2012

Self Cite

View full text Add to dashboard Cite

The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. This project focuses on the development of highly effective non-chromatographic methods for purifying recombinant proteins and enzymes. This report covers work at Ohio State University, which focuses on the further optimization of the purification technology in the PI's new laboratory. A key aspect of the technology involves the use of a self-cleaving protein module, which can be used to make any purification tag self-cleaving. By combining this module with a variety of purification tags, we have generated a number of simple, inexpensive and highly ABSTRACTThis project focuses on the development of highly effective non-chromatographic methods for purifying recombinant proteins and enzymes. This report covers work at Ohio State University, which focuses on the further optimization of the purification technology in the PI's new laboratory. A key aspect of the technology involves the use of a self-cleaving protein module, which can be used to make any purification tag self-cleaving. By combining this module with a variety of purification tags, we have generated a number of simple, inexpensive and highly effective methods for protein purification. We have applied these methods to the production of several proteins, including organophosphohydrolase (OPH), which can be used to degrade nerve agents and environmentally persistent insecticides. We have further demonstrated these methods in high cell-density fermentation at laboratory scale, and have provided evidence of their effectiveness. Our most recent work has been on the optimization of the fermentation process itself, as well as a more biochemical optimization of the expression system. Overall, the ARO support on this project has yielded two patent applications, seven peer-reviewed papers and one book chapter. In addition to these, three manuscripts are in late stages of preparation.

show abstract

Towards the elimination of chromatography in protein purification: Expressing proteins engineered to purify themselves

Cited by 16 publications

References 47 publications

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

Active tyrosine phenol-lyase aggregates induced by terminally attached functional peptides in Escherichia coli

Production of Self-Purifying Proteins in a Variety of Expression Hosts with Focus on Organophosphorus Hydrolase

Contact Info

Product

Resources

About