Nicolas Abello | PubMed Health

PubMed2006

Study of human lung elastin degradation by different elastases using high-performance liquid chromatography/mass spectrometry.

Elastin is a structural insoluble protein which gives elasticity to tissues and organs. Although its hydrophobic and highly cross-linked nature makes it a very durable polymer, degradation of elastin in relation with several pathological conditions, such as pulmonary emphysema, has been documented. Since different enzymes may be involved in elastolysis, it is of interest to determine which enzyme is responsible for the degradative effects observed in a certain disease. The aim of this work was to study elastin degradation by proteases from different families (serine, cysteine, and metalloproteases) using liquid chromatography coupled to mass spectrometry to characterize the elastin-derived peptides. Incubation of insoluble human elastin with different elastases revealed that, indeed, each protease degrades elastin in a preferential way giving rise to specific peptide patterns. This opens the possibility of using a given set of peptides as biomarkers for disease-related elastolysis.

Amino Acid Sequence↗

PubMedDate not supplied

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins.

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.

Blotting, Western↗