His-tag β-galactosidase supramolecular performance

Flores, Sandra S.; Clop, Pedro D.; Barra, José L.; Argaraña, Carlos E.; Perillo, Marı́a A.; Nolan, Verónica; Sánchez, Julieta M.

doi:10.1016/j.bpc.2021.106739

Cited by 5 publications

(2 citation statements)

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“…Figure 3a shows the pull-down result of LacZ. As demonstrated in the figure, LacZ was expressed as IBs using LHS1, 0.4 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) for 20 h at 15-20 • C. LacZ monomer has 116 kDa [33]. To obtain active LacZ inclusion bodies, we used the short tag LHS1.…”

Section: Resultsmentioning

confidence: 99%

Physiologically Aggregated LacZ Applied in Trehalose Galactosylation in a Recycled Batch Mode

Belkova,

Janegova,

Hrabarova

et al. 2023

Life

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Galactooligosaccharides obtained via β-galactosidase transgalactosylation have health-promoting properties and are widely recognized as effective prebiotics. Trehalose-based galactooligosaccharides could be introduced into food and pharmaceutical industries similarly to trehalose. In light of this, new technological approaches are needed. Recently, in vivo enzyme immobilizations for recombinant proteins have been introduced, and physiological aggregation into active inclusion bodies (aIBs) has emerged as one such method of in vivo immobilization. To prepare LacZ β-galactosidase in the form of aIBs, we used a short 10 amino acid aggregation-prone tag. These native protein particles were simply washed from the cell lysate and applied in trehalose galactosylation in a recycled batch mode. In this study, aIBs entrapped in alginate beads, encapsulated in alginate/cellulose sulfate/poly(methylene-co-guanidine) capsules and magnetized were compared with free aIBs. Alginate/cellulose sulfate/PMCG capsules showed more suitable properties and applicability for biotransformation of trehalose at its high concentration (25%, w/v) and elevated temperature (50 °C).

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Section: Resultsmentioning

confidence: 99%

Physiologically Aggregated LacZ Applied in Trehalose Galactosylation in a Recycled Batch Mode

Belkova,

Janegova,

Hrabarova

et al. 2023

Life

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“…His-tags are convenient: being short, they allow elution in mild native conditions and provide at the same time a reasonable degree of purity. But in some cases placing His-tags at protein’s termini may create problems: a classical hexahistidine sequence can cause protein inclusion body formation , or oligomerization through histidine residues, , can impair catalytic enzyme functions − or protein–protein interaction, or His-tags cannot be used at N- or C-termini because of their functional and/or structural importance. In these cases, His-tags could be used in a noncanonical way by inserting them into the polypeptide chain.…”

Section: Introductionmentioning

confidence: 99%

Novel His-tag Variants for Insertion Inside Polypeptide Chain

Tarabarova,

Lopukhov,

Fedorov

et al. 2023

ACS Omega

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His-tags are protein affinity tags ubiquitously used due to their convenience and effectiveness. However, in some individual cases, the attachment of His-tags to a protein's N-or Ctermini resulted in impairment of the protein's structure or function, which led to attempts to include His-tags inside of polypeptide chains. In this work, we describe newly designed internal His-tags, where two triplets of histidine residues are separated by glycine residues to avoid steric hindrances and consequently minimize their impact on the protein structure. The applicability of these His-tags was tested with eGFP, a multifaceted reference protein, and GrAD207, a modified apical domain of GroEL chaperone, designed to stabilize in soluble form initially insoluble proteins. Both proteins are used as fusion partners for different purposes, and providing them with His-tags introduced into their polypeptide chains should conveniently broaden their functionality without involving the termini. We conclude that the insertable tags may be adjusted for the purification of proteins belonging to different structural classes.

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