Expression, Single-Step Purification, and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific β-Glucosidase

Zouhar, Jan; Nanak, Elizabeth; Brzobohatý, Břetislav

doi:10.1006/prep.1999.1108

Cited by 44 publications

(25 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Site-directed mutagenesis of (His) 6 Zm-p60.r was performed using the GeneEditor TM in vitro site-directed mutagenesis system on plasmid pRSET::Zm-p60.r [15]. Briefly, pRSET::Zm-p60.r allows expression of a Zm-p60.1 coding region corresponding to the processed mature Zm-p60.1 polypeptide (starting with a serine residue in position 60 predicted from the full size Zm-p60.1 cDNA) fused to a His-tag.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Rotrekl¹,

Nejedlá²,

Kučera³

et al. 1999

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The maize Zm-p60.1 gene encodes a b-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site-directed mutagenesis, and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea-and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the K m values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme±substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k H cat . C170 is located on a hydrophobic side of an a-helix packed against hydrophobic aminoacid residues of b-strand 4, indicating participation of C170 in stabilization of a (b/a) 8 barrel structure in the enzyme. In C479A/D/R/S mutants, K m and k H cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.Keywords: b-glucosidase; cysteine residues; disulfide bridge; structure±fuction relationships.Current research on b-glucosidases of animals, plants and microorganisms has significant scientific, clinical and economic implications (reviewed in [1]). In plants, b-glucosidases have been implicated in various aspects of growth, productivity and defence, and reactions such as cyanogenesis related to food and feed toxicity. In addition, recent data indicate that plant b-glucosidases may be involved in the metabolism of plant hormones, whose storage forms occur as b-glucosides and are activated by b-glucosidases [2,3]. Thus, information on structure±function relationships in the b-glucosidases is of obvious importance for understanding their biological functions, and engineering their catalytic and molecular properties for industrial and field applications.b-Glucosidases catalyse the hydrolysis of aryl and alkyl-b-dglucosides as well as glucosides with only carbohydrate moieties (such as cellobiose). Catalysis involves two essential carboxylates, one acting as a proton donor and the other as a nucleophile [4]. Cleavage of the glucosidic bond results in retention of the configuration at the anomeric carbon atom.Plant b-glucosidases are classified into family 1 of glycos...

show abstract

Section: Site-directed Mutagenesismentioning

confidence: 99%

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Rotrekl¹,

Nejedlá²,

Kučera³

et al. 1999

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…ing overcomes this problem, as the matrix-bound protein is prevented from interacting with surrounding ~ 4oooo protein molecules and the d e n a t u r a n t can be completely ~= removed, thereby preventing aggregation (17)(18)(19)(20). We 20000 have overexpressed and refolded histidine sensor kinase DevS of M. tuberculosis using a matrix-assisted o refolding protocol.…”

Section: Discussionmentioning

confidence: 97%

“…Recent advances in protein folding have led to the development of matrix-bound renaturation techniques which could be very effective in folding proteins not amenable to conventional refolding protocols (7,8). Solubilization of proteins on a solid matrix was previously reported by many groups on a variety of matrices including heparin-Sepharose, cellulose (20), and Ni2+-NTA, among others (10,(17)(18)(19). Ni2+-NTA resin is a matrix of choice since it is based on the specific affinity of Ni2+-NTA to 6× His and allows conformation-independent binding of tagged proteins.…”

Section: Discussionmentioning

confidence: 97%

Cloning, Overexpression, Purification, and Matrix-Assisted Refolding of DevS (Rv 3132c) Histidine Protein Kinase of Mycobacterium tuberculosis

Saini¹,

Pant²,

Das

et al. 2002

Protein Expression and Purification

View full text Add to dashboard Cite

“…Refolding of fusion chitinase was carried out in the Ni 2+ -NTA column (Zouhar et al 1999). The refolding process was started with 10 column volumes of RB 1 , followed by 15 column volumes of RB 2 .…”

Section: Denaturation and Refolding Of Chitinasementioning

confidence: 99%

Polyclonal antibody against Manduca sexta chitinase and detection of chitinase expressed in transgenic cotton

et al. 2005

View full text Add to dashboard Cite

The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni2+-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.

show abstract

Expression, Single-Step Purification, and Matrix-Assisted Refolding of a Maize Cytokinin Glucoside-Specific β-Glucosidase

Cited by 44 publications

References 18 publications

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Cloning, Overexpression, Purification, and Matrix-Assisted Refolding of DevS (Rv 3132c) Histidine Protein Kinase of Mycobacterium tuberculosis

Polyclonal antibody against Manduca sexta chitinase and detection of chitinase expressed in transgenic cotton

Contact Info

Product

Resources

About