High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC)

Camilo, César M.; Polikarpov, Igor

doi:10.1016/j.pep.2014.03.008

Cited by 52 publications

(39 citation statements)

References 56 publications

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“…The gene encoding Bl Cel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Bl cel5B_Fw and Bl cel5B_Rv ( Supplementary Table 3 ). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere 30 .…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Liberato

Silveira

Prates

et al. 2016

Sci Rep

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Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

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Section: Methodsmentioning

confidence: 99%

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Liberato

Silveira

Prates

et al. 2016

Sci Rep

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“…César Moisés Camilo cloned the gen BAD_1325 in the vector pET-TrxA using the method Ligation-Independent Cloning (LIC) and screened solubility in a semi-automated way in E. coli. 56 12 enzymes were cloned, including the gen BAD-1325, without the need of restriction enzymes (methodology in more detail in reference 54). 56 The ORF of gen…”

Section: Organism Media Plasmids and Culture Conditionsmentioning

confidence: 99%

“…56 12 enzymes were cloned, including the gen BAD-1325, without the need of restriction enzymes (methodology in more detail in reference 54). 56 The ORF of gen…”

Section: Organism Media Plasmids and Culture Conditionsmentioning

confidence: 99%

Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

Mera¹

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MONSALVE ALAIN, M. Structural and enzymatic features of a recombinant βfructofuranosidase from Bifidobacterium adolescentis. 2016. 92 p. Dissertation (Master in Science) -Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, 2016.Despite the fact that Glycosyl Hydrolase Family 32 present 4 467 enzyme entries, only 14 of them have been characterized structurally. From the ten protein crystal structures deposited for Bifidobacterium adolescentis ATCC 15703 at PDB just one enzyme is related to the processing of non-digestible sugars and there is no structure of a β-fructofuranosidase. In this research we studied the biochemical properties and the structural features of a recombinant βfructofuranosidase (BaFFse) from the healthy gut bacteria B. adolescentis ATCC 15703 (gen BAD_1325) heterologously expressed in Escherichia coli Rosetta. The enzyme was purified by nickel ion affinity chromatography and molecular exclusion chromatography; the purification process was judged by denaturing SDS-PAGE gel. Sucrose was used as a substrate for the enzyme activity assays and the amount of reducing sugars, detected by Dinitrosalycilic acid, was taken as indicator of the optimum conditions of hydrolysis for the enzyme. BaFFase crystal, grown in PEG 8K 25% (w/v) and buffer MES 0.1M pH 6.5, was diffracted at 2.44 Å and processed using the CCP4 program package. The enzyme presented a classical four-stranded five-bladed β-propeller and a C-terminal β-sandwich characteristic from the GH 32 family; however, connected to the β-propeller through a loop of 38 residues, BaFFase also presented an N-terminal β-sandwich domain, which sequence (residues 3-100 from BaFFase) did not match with any protein sequence when aligned against PDB database.Assays with Gel filtration calibration, DLS and SAXS showed that the enzyme was a stable homodimer in solution. Based on the superposition of structures using the a βfructofuranosidase from B. longum KN29.1 we could deduced the three key aminoacids involved in the transferring of fructosyl moieties by BaFFase. A nucleophile attack is performed by the carboxylate of Asp 131, forming the fructose -BaFFase intermediate; Glu 375 donates a proton, acting as an acid base catalyst and Asp 269 stabilizes the transitions state in the fructosyl transferring activity. This is the first GH32 oligomeric enzyme belonging to the bacteria kingdom. We have described a novel additional β-sandwich domain for a GH32 enzyme that increases the region of contact to form a dimer. This is the first βfructofuranosidase crystal structure from the microorganism B. adolescentis ATCC 15703.

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“…While there are many semiautomated pipelines available for cloning, expression and purification, there are hardly any pipelines that involve complete automation of these steps; that is individual steps such as PCR set‐up, production, recovery of biomass, purification of DNA and proteins are automated, but certain steps such as transformation step and DNA and protein gel analysis in most pipelines involve some amount of manual intervention. For example, the spreading on LB agar plates is performed by twisting the plates manually after addition of culture solution (Camilo and Polikarpov, ). Also, the lid of the agar plate has to be closed manually after drying of the plate is achieved.…”

Section: Introductionmentioning

confidence: 99%

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies

Bairy

Gopalan

Setty

et al. 2018

Microbial Biotechnology

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SummaryThe process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His‐tagged Gateway vector, followed by their small‐scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large‐scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

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High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC)

Cited by 52 publications

References 56 publications

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies

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High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC)

Cited by 52 publications

References 56 publications

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Structural and enzymatic features of a recombinant &beta;-fructofuranosidase from <i>Bifidobacterium adolescentis</i>

Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies

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Structural and enzymatic features of a recombinant β-fructofuranosidase from <i>Bifidobacterium adolescentis</i>