Juan J Calvete — Page 4

PubMed2002

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells.

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.

Blotting, Western↗

PubMed2001

Crystallization and preliminary X-ray diffraction analysis of the seed lectin from Parkia platycephala.

The crystallization and preliminary X-ray diffraction analysis of the seed lectin of Parkia platycephala, a Mimosoideae, regarded as the most primitive group of the Leguminosae plants, are reported. Its amino-acid sequence consists of three tandemly arranged jacalin-related beta-prism domains, which is a novel fold for a leguminous lectin. Furthermore, no other lectin structure with this arrangement of domains has been described. P2(1)2(1)2(1) crystals (unit-cell parameters a = 63.6, b = 68.5, c = 208.5 A), which diffract to a maximum resolution of 2.2 A, were obtained in hanging drops at pH 8 and 293 K by the vapor-diffusion method using 10% 2-propanol and 20% polyethylene glycol 4000 as precipitants. The asymmetric unit contains two lectin molecules and has a solvent content of 46%. Only a single beta-prism domain could be located by molecular replacement using the structure of the Helianthus tuberosus lectin (PDB code 1c3k) as the search model. Isomorphous heavy-atom derivatives are currently being produced to solve the complete structure of the P. platycephala seed lectin.

Crystallization↗

PubMedDate not supplied

Does seminal plasma PSP-I/PSP-II spermadhesin modulate the ability of boar spermatozoa to penetrate homologous oocytes in vitro?

Low concentration (0.15 mg per million of spermatozoa) of seminal plasma-derived PSP-I/PSP-II spermadhesin heterodimer is able to preserve the viability of highly extended boar spermatozoa. Whether spermatozoa also keep their fertilizing capacity is not yet known. The present study evaluated the effect of exposing freshly extended and frozen-thawed boar spermatozoa (10 million/mL) to PSP-I/PSP-II (1.5 mg/mL) for 30 or 120 minutes on sperm characteristics and the outcome of in vitro penetration of immature (IM) and in vitro matured (IVM) homologous oocytes, aiming to identify this spermadhesin as a suitable modulator for sperm-handling protocols. Although exposure to the heterodimer improved sperm viability and motility without increasing the levels of sperm acrosome exocytosis in both freshly extended and frozen-thawed spermatozoa, this pretreatment did not affect sperm penetration rates or sperm numbers per oocyte when pretreated fresh spermatozoa were coincubated with IM or IVM oocytes compared with controls. When cryopreserved spermatozoa were tested, however, on IVM oocytes, already a 30-minute preincubation exposure to PSP-I/PSP-II showed a significant blocking effect on penetration rate (from 90% to 32%, P < .05) and on mean sperm numbers per oocyte (2.9 to 1.6, P < .05). To disclose the nature of this paradox, frozen-thawed spermatozoa were cleansed (by centrifugation in saline bovine serum albumin or through Percoll density gradient separation) and the procedure repeated. Oocyte penetration (but not number of spermatozoa per oocyte) increased (P < .05) when spermatozoa were cleansed with Percoll compared with either washed or unwashed controls (53% vs 13% vs 31%, respectively). In addition, the percentages of polyspermic oocytes remained lower than control (38.5% vs 68.7%, respectively; P < .05). In conclusion, the results confirm that exposure of fresh or frozen-thawed boar spermatozoa to a low dose of seminal PSP-I/PSP-II spermadhesin preserves sperm viability and motility in vitro. Although there was no obvious influence of the heterodimer on the capability of freshly extended boar spermatozoa to penetrate homologous oocytes (either IM or IVM), PSP-I/PSP-II exerted a deleterious effect when frozen-thawed spermatozoa were used to penetrate IVM oocytes. Such an effect of cryopreservation seems to a certain extent reversible, since cleansing of the sperm surface decreased, at least partially, this blocking effect, increasing both penetration and the monospermic rates.

Animals↗