Carbohydrate-binding protein 35: molecular cloning and expression of a recombinant polypeptide with lectin activity in Escherichia coli.

Affinity-purified antibodies directed against carbohydrate-binding protein 35 (CBP35), a galactose-specific lectin, were used to screen a lambda gt 11 expression library derived from mRNA of 3T3 fibroblasts. This screening yielded several putative clones containing cDNA for CBP35, one of which was characterized in terms of its expression of a fusion protein containing beta-galactosidase and CBP35 sequences. Limited proteolysis of lysates containing the fusion protein, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-CBP35, yielded a peptide mapping pattern comparable to that obtained from parallel treatment of authentic CBP35. Such a limited proteolysis followed by affinity chromatography on a Sepharose column coupled with galactose also yielded a 30-kDa polypeptide that exhibited carbohydrate-binding activity. This polypeptide can be immunoblotted with anti-CBP35, but not with antibodies directed against beta-galactosidase. These results indicate that we have identified a cDNA clone for CBP35 that yields a recombinant polypeptide with lectin activity produced in Escherichia coli. Using this cDNA clone as a probe, Northern-blot analysis showed an increased expression of the CBP35 gene when quiescent 3T3 cells were activated by the addition of serum growth factors.

Design of active analogues of a 15-residue peptide using D-optimal design, QSAR and a combinatorial search algorithm.

This report describes the rational design of novel analogues of a 15-residue antibacterial peptide CAMEL0. A constrained D-optimal design was carried out to derive a training set of 60 analogues. Partial least squares (PLS) models describing quantitative structure-activity relationships (QSARs) were initially derived for the peptides using two published and one novel parameter set. The novel "Design parameters' were based on key structural features identified in hypothetical models of the mechanisms by which peptides interact with cell membranes. In an extension of the PLS method, influence statistics were used to decrease the weighting of compounds having a large effect on model predictions. A combinatorial search algorithm was developed which used PLS models as predictors to select a test set of 39 peptides with high predicted potencies. Within this set, the most potent analogue CAMEL135, which contained seven point mutations from CAMEL0, was identified. For a panel of 24 bacteria, the mean MIC value of CAMEL135 was approximately half of that for CAMEL0. For the parameter sets tested, covariance functions derived from Z-scales gave highest Q2-values for the training set, whilst the model using the the 'Design parameters' gave least error when predicting the activity of the test set. The predictive ability of a third published set of peptide parameters was found to compare favourably with that of the parameters used in the design. Analysis of the PLS models indicates that hydrophobicity and amphipathicity are the most important features influencing activity for this class of compound.

Empirical scoring functions: I. The development of a fast empirical scoring function to estimate the binding affinity of ligands in receptor complexes.

This paper describes the development of a simple empirical scoring function designed to estimate the free energy of binding for a protein-ligand complex when the 3D structure of the complex is known or can be approximated. The function uses simple contact terms to estimate lipophilic and metal-ligand binding contributions, a simple explicit form for hydrogen bonds and a term which penalises flexibility. The coefficients of each term are obtained using a regression based on 82 ligand-receptor complexes for which the binding affinity is known. The function reproduces the binding affinity of the complexes with a cross-validated error of 8.68 kJ/mol. Tests on internal consistency indicate that the coefficients obtained are stable to changes in the composition of the training set. The function is also tested on two test sets containing a further 20 and 10 complexes, respectively. The deficiencies of this type of function are discussed and it is compared to approaches by other workers.