An active single-chain antibody containing a cellulase linker domain is secreted by <i>Escherichia coli</i>

Takkinen, Kristiina; Laukkanen, Marja−Leena; Sizmann, Dorothea; Alfthan, Kaija; Immonen, Tiina; Vanne, Liisa; Kaartinen, Maija; Knowles, Jonathan; Teeri, Tuula T.

doi:10.1093/protein/4.7.837

Cited by 104 publications

(51 citation statements)

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“…Standard protocols were used for all DNA manipulations (23). In brief, the expression plasmid was based on the vector pKK223-3 with the tac promoter and the pelB signal sequence of Erwinia carotovora (24). The expression cassette contained the cDNA segments coding for the 38 amino acids of the CBHII CBD, 3 amino acids of the CBHII linker (plasmid pTTc9) (25), 21 residues of the CBHI linker, and finally the 36 residues of the-CBHI CBD (plasmid pTTcl) (26).…”

Section: Methodsmentioning

confidence: 99%

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Linder

Teeri

1996

Proc. Natl. Acad. Sci. U.S.A.

165

126

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Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H.Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.Cellulose is a polymer composed of D-glucose units linked by 13-1,4-glycosidic bonds. The cellulose chains can pack together to form fibers, which are crystalline in nature (1). This solid and ordered substrate poses some special requirements on the enzymes hydrolyzing it. Most cellulose degrading enzymes have a bifunctional organization composed of Iwo domains connected by a linker region. One of the domaind is dedicated for binding to the solid cellulose and is called a cellulosebinding domain (CBD). The other domain contains the catalytic machinery. These catalytic domains have been grouped into several families sharing the same basic structure and the same chemical reaction mechanism (2). Trichoderma reesei cellobiohydrolase I (CBHI) is an exoglucanase with a long tunnel-shaped active site (3). CBHI and other exoglucanases initiate the hydrolysis at the chain ends, and do not produce significant amounts of new chain ends on the cellulose surface (4). They seem to act by a processive mechanism hydrolyzing consecutive bonds along the same chain without dissociating (2, 3). The endoglucanases cleave internal bonds along the cellulose chains probably by cycles of adsorption and desorption.Based on sequence comparisons the CBDs have been grouped into several families (5). To date, the threedimensional structures of two CBDs have been published. One is from T. reesei CBHI (6) and the other from Cellulomonas fimi xylanase/cellobiohydrolase Cex (7). Both the fold and the size of these CBDs are different, but a common pattern of aromatic residues and amides is found at the surface of both proteins. Many experiments have shown that this surface is the primary interaction site between a CBD and cellulose (7-10). Many studies directly correlate the efficiency of crystalline cellulose degradation to the binding efficiency of the enzyme (11) and the removal of a CBD typically results in a decrease of a...

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Section: Methodsmentioning

confidence: 99%

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Linder

Teeri

1996

Proc. Natl. Acad. Sci. U.S.A.

165

126

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“…Six histidine residues were cloned to the C-terminus of the heavy chain for purification and immobilization purposes. Histidine-tagged Fab fragment was cloned into the pKKtac expression vector [38] for large-scale production of soluble Fab fragments in E. coli strain RV308 (ATCC 31608). Highcell-density fermentation (fed-batch fermentation (4.5 L) in a Bio-Flow IV fermenter (New Brunswick)) and purification by metal affinity (copper-IMAC) and protein G chromatography (Amersham-Pharmacia) were done as described by Nevanen et al [39].…”

Section: Antitestosterone Fab Fragmentmentioning

confidence: 99%

Microscale immunoaffinity SPE and MEKC in fast determination of testosterone in male urine

et al. 2007

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Conventional methods for the determination of testosterone in body fluids typically suffer from poor recovery, lack of specificity, complex sample pretreatment, or the need for derivatization. Here, a simple, specific, and fast analysis method for testosterone was developed, with a methodology based on testosterone-specific immunoaffinity SPE (IA-SPE) and subsequent analysis by partial filling MEKC (PF-MEKC). An immunosorbent consisting of a recombinant antitestosterone Fab fragment covalently attached to activated Sepharose was prepared. IA-SPE and PF-MEKC were set up in hyphenated and off-line constructions, and the applicability of the two constructions in analysis of testosterone in male urine was investigated. The results obtained with the hyphenated construction proved to be only indicative of the presence of testosterone. The off-line IA-SPE and PF-MEKC construction, however, was successfully used in the determination of free testosterone in male urine samples after enzymatic hydrolysis of the glucuronide conjugates. Except for the hydrolysis reaction, no sample pretreatment was required. After hydrolysis, the overall analysis time per sample was only 14 min. The off-line IA-SPE and PF-MEKC method proved to be robust, sensitive (LOQ 35 mg/L), and specific, enabling separation of testosterone from four related steroids. Thus, it provides attractive features when compared to traditional methods for determination of testosterone in male urine.

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“…The most promising clones, which had decreased DHEAS cross-reactivity and/or improved relative testosterone binding affinity when compared with the clone A60/HCDR1/LCDR2, were sequenced, cloned into the pKKtac expression vector (15), and transformed into the Escherichia coli strain RV308 for small-scale production. Clones were characterized using goat anti-mouse IgG-coated microtiter wells and testosterone-3-CMO-polylysine labeled with fluorescent Europium-chelate (Wallac) as the label.…”

Section: Table II Affinities and Cross-reactivities Of The Mutant Antmentioning

confidence: 99%

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

Valjakka

Hemminki

Niemi

et al. 2002

Journal of Biological Chemistry

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A highly selective, high affinity recombinant anti-testosterone Fab fragment has been generated by stepwise optimization of the complementarity-determining regions (CDRs) by random mutagenesis and phage display selection of a monoclonal antibody (3-C 4 F 5 ). The best mutant (77 Fab) was obtained by evaluating the additivity effects of different independently selected CDR mutations. The 77 Fab contains 20 mutations and has about 40-fold increased affinity (K d ‫؍‬ 3 ؋ 10 ؊10 M) when compared with the wild-type (3-C 4 F 5 ) Fab. To obtain structural insight into factors, which are needed to improve binding properties, we have determined the crystal structures of the mutant 77 Fab fragment with (2.15 Å) and without testosterone (2.10 Å) and compared these with previously determined wild-type structures. The overall testosterone binding of the 77 Fab is similar to that of the wild-type. The improved affinity and specificity of the 77 Fab fragment are due to more comprehensive packing of the testosterone with the protein, which is the result of small structural changes within the variable domains. Only one important binding site residue Glu-95 of the heavy chain CDR3 is mutated to alanine in the 77 Fab fragment. This mutation, originally selected from the phage library based on improved specificity, provides more free space for the testosterone D-ring. The light chain CDR1 of 77 Fab containing eight mutations has the most significant effect on the improved affinity, although it has no direct contact with the testosterone. The mutations of CDR-L1 cause a rearrangement in its conformation, leading to an overall fine reshaping of the binding site.Steroid hormones have remained a great challenge for immunodiagnostics. They are small, rigid, hydrophobic molecules with only a few functional groups capable of specific interactions with antibodies. The number of different closely related steroids in human serum is high, their in vivo concentrations are low (down to a picomolar level), and their relative concentrations can vary greatly, even between normal healthy individuals. Furthermore, steroids are poorly immunogenic in mice and rats; two species from which monoclonal antibodies are usually generated, making it extremely difficult to produce high affinity and specificity anti-steroid monoclonal antibodies. In most diagnostic immunoassays of steroid hormones, rabbit polyclonal antibodies are used, despite the many drawbacks associated with the use of antisera. The supply of good polyclonal antibody reagents of uniform quality is a severe problem for the immunodiagnostic industry and requires continuous immunization of many laboratory animals.Antibody engineering provides excellent tools to tailor the properties of antibodies with respect to affinity, specificity, and performance for different applications. An in vitro process of antigen-driven selection, based on the display of antibody fragments on the surface of a filamentous bacteriophage, has been shown to be a powerful method for the selection of specific or improved...

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An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Cited by 104 publications

References 0 publications

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Microscale immunoaffinity SPE and MEKC in fast determination of testosterone in male urine

Crystal Structure of an in Vitro Affinity- and Specificity-matured Anti-testosterone Fab in Complex with Testosterone

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