Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails

Lilius, Gösta; Persson, Mats; Bülow, Leif; Mosbach, Klaus

doi:10.1111/j.1432-1033.1991.tb16042.x

Cited by 85 publications

(30 citation statements)

References 27 publications

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“…zinc in kininogen and heme iron in the plasmodial protein and HRG. Moreover, His-rich nereid jaw proteins are high in complexed copper or zinc (27), and poly-(His) tracts are routinely added to recombinant proteins for facile recovery by metalloprecipitation or metalloligand affinity chromatography (28,29). Of the transition metals detected in M. edulis byssus, zinc levels are consistently the highest at about 1 mg/g of dry weight (30).…”

Section: Resultsmentioning

confidence: 99%

Tough Tendons

Xx¹,

Coyne²,

Jh³

1997

Journal of Biological Chemistry

136

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The primary structure of the ␣-chain of preCol-D (molecular mass ‫؍‬ 80 kDa), a tanned collagenous protein predominating in the distal portion of the byssal threads of the mussel Mytilus edulis, was deduced from cDNA to encode an unprecedented natural block copolymer with three major domain types: a central collagen domain flanked by fibroin-like domains and followed by histidine-rich termini. The fibroin-like domains have sequence motifs that strongly resemble the crystalline polyalanine-rich and amorphous glycine-rich regions of spider dragline silk fibroins. The terminal regions resemble the histidine-rich domains of a variety of metalbinding proteins. The silk domains may toughen the collagen by increasing its strength and extensibility. PreCol-D expression is limited to the mussel foot, which contains a longitudinal gradient of preCol-D mRNA. This gradient increases linearly in the proximal to distal direction and reaches a maximum just before the distal depression of the foot.Marine mussels Mytilus edulis produce byssal threads to attach to solid surfaces in the turbulent intertidal zone. Each thread is stiff at one end and extensible at the other. Molecular gradients correlated with a stiff-to-extensible mechanical gradation exist along the length of each thread (1, 2). Byssal threads (Fig. 1A) are often compared with tendons on the basis of their construction from anisotropically oriented bundles of collagen fibrils (3). Indeed, the distal portion of byssus and tendon share a roughly similar tensile strength and initial stiffness (4), but the similarity ends there. Byssal threads are at least five times more extensible and five times tougher than Achilles tendon (5). Moreover, unlike tendon, byssal threads have a nonperiodic microstructure and shrinkage and melting temperatures in excess of 90°C (6). Understanding how byssal collagen differs from other collagens is a critical first step to appreciating its role in the material performance of byssus.The characterization of byssal collagens has long been thwarted by the intractable cross-linked nature of byssus. Two independent lines of evidence, however, have hinted at an unusual possibility: that byssal collagen may be endowed with some qualities of a silk fibroin-like structure. The first evidence is based on wide-angle fiber x-ray diffraction of byssal threads of Mytilus edulis. The distal part consistently exhibits a typical collagen diffraction pattern with the notable addition of strong equatorial reflections at 4.5-4.6 Å and nonaxial arcs at 3.7-3.8 Å (2, 7). These prompted Rudall (7) to suggest the presence of a ␤-phase in the byssal collagen or an additional ␤-protein in the distal portion of byssus.In the second line of evidence, byssal threads of M. edulis were subjected to acid extraction coupled with extensive pepsinization. This approach solubilized two collagenous fragments, Col-P 1 and Col-D (both apparently homotrimers), predominating at the proximal and distal end of each thread, respectively (1). Precursors of these fragments, preCol-P ...

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Section: Resultsmentioning

confidence: 99%

Tough Tendons

Xx¹,

Coyne²,

Jh³

1997

Journal of Biological Chemistry

136

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“…Plausible models reconciling axial sequence with the stochastic distribution of preCols in byssal threads can be contrived for almost any combination of homo-and/or copolymer chains in which spatial variability is controllable. Given the metal-induced 'stickiness' of the histidine-rich ends, even short chains would readily combine into longer ones upon the addition of Cu and/or Zn (see, for example, Lilius et al 1991).…”

Section: Assembly and Cross-linkingmentioning

confidence: 99%

Elastomeric gradients: a hedge against stress concentration in marine holdfasts?

Waite

Vaccaro

Sun

et al. 2002

Phil. Trans. R. Soc. Lond. B

119

138

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The byssal threads of marine mussels are elastomeric fibres with a great capacity for absorbing and dissipating energy. Up to 70% of the total absorbed energy can be dissipated in the byssus. Because byssal threads attach the mussel to hard inert surfaces in its habitat, they must combine the need to be good shock absorbers with appropriate matching of Young's modulus between living tissue and a hard substratum such as stone-stiffnesses that can differ by five orders of magnitude. Recent data suggest that improved modulus matching and decreased stress concentration between different portions of the byssus is achieved by the use of protein gradients. Protein gradients in byssal threads are constructed using natural macromolecular chimeras having a central collagenous domain, variable flanking modules and histidine-rich amino and carboxy termini. Stiff silk-like flanking modules prevail distally, while at the animal end, rubbery modules resembling elastin predominate. In between the two thread ends there is a mix of both module types. The histidine-rich termini provide metal binding/cross-linking sites, while collagen domains may confer self-assembly on all parts of the structure. A graded axial distribution of flanking modules is expected to moderate stress concentration in joined materials having disparate moduli.

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“…The introduction of (poly)histidine tags to the N-and C-termini of proteins via genetic manipulation has greatly simplified their purification through affinity chromatography on metal chelate columns (1)(2)(3). As well as aiding in the purification of proteins, introduced "bio-tags" have been shown to aid the oriented immobilization of proteins on surfaces in order to retain specific activities and prevent denaturation (4 -6).…”

mentioning

confidence: 99%

“…L-Lactate dehydrogenase (L-LDH) 2 from Bacillus stearothermophilus is an NAD(H)-dependent oxi-doreductase that catalyzes the interconversion of Llactate and pyruvate (11). The enzyme exists as an allosteric tetramer, composed of four identical subunits, each of relative molecular mass of approximately 35,000 (12).…”

mentioning

confidence: 99%