The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins

Gräslund, S.; Sagemark, Johanna; Berglund, H.; Dahlgren, Lars-Göran; Flores, Alex F. C.; Hammarstrom, M.; Johansson, Ida; Kotenyova, T.; Nilsson, M.E.; Nordlund, P.; Weigelt, J.

doi:10.1016/j.pep.2007.11.008

Cited by 113 publications

(105 citation statements)

References 30 publications

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“…35 It has been noted that single domains of proteins are more likely to be successfully expressed in E. coli than large, multidomain proteins. 36 This observation is consistent with genetic construct design strategies applied to TSOL18 and the toward conformational epitopes. These findings indicated that since the host protective epitopes of TSOL18 appear to be conformational, options for the development of a vaccine based on defined epitopes produced as synthetic peptides are limited.…”

Section: Vaccine Development Against the Taenia Solium Parasitesupporting

confidence: 78%

Vaccine development against theTaenia soliumparasite

2013

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Section: Vaccine Development Against the Taenia Solium Parasitesupporting

confidence: 78%

Vaccine development against theTaenia soliumparasite

2013

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“…Protein crystallographers, who need to crystallize proteins, commonly adopt multiconstruct strategies, where many different constructs of the same protein are used by lengthening/ shortening the amino acid sequence. 3,4 Many crystallographers remember some anecdotic case in which well diffracting crystals were obtained by removing/adding just one or two residues at the sequence termini. These ''miracles'' can have various interpretations.…”

Section: Introductionmentioning

confidence: 99%

Participation of protein sequence termini in crystal contacts

Carugo

2011

Protein Science

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The analysis of the crystal packing interactions, in a nonredundant set of high resolution and monomeric globular protein crystal structures, shows that the residues located at the N-and C-termini of the sequence tend to participate in packing interaction more often than expected and that often they interact with each other. Since the sequence termini are, in general, conformationally very flexible and since they host electrical charges of opposite sign, it can be hypothesized that they play a crucial role in the early formation of the nonphysiological contacts that bring to protein crystallization. It is thus not surprising that modest lengthening/shortening of the sequence termini have often a dramatic effect on protein crystallogenesis.

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“…Potential variables to be optimized include, but are not limited to, varying expression strains 1,2 , temperature 3,4 , media 2,3 , target variants 5 , fusion partners [6][7][8][9][10][11][12][13] , co-expression with chaperones 14,15 , cytoplasmic or periplasmic expression [16][17][18] , and purification buffer components 3 . By implementing high throughput approaches, many variables or many targets can be tested in parallel with a high level of efficiency, while limiting batch-to-batch variation.…”

Section: Introductionmentioning

confidence: 99%

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

Saez

Nozach

Blémont

et al. 2014

JoVE

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Escherichia coli ( E. coli ) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4 ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment. Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project ( www.venomics.eu ), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

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The use of systematic N- and C-terminal deletions to promote production and structural studies of recombinant proteins

Cited by 113 publications

References 30 publications

Vaccine development against theTaenia soliumparasite

Vaccine development against theTaenia soliumparasite

Participation of protein sequence termini in crystal contacts

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

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