Jérôme Becquart

High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1β

Abstract A high-productivity process has been developed for the production of mature human interleukin-1β (IL-1β) from recombinant Escherichia coli strains. Conditions were found that allow high IL-1β expression levels in high cell density cultures. Improved fed-batch fermentation strategies are described which include maintenance of glucose and acetate concentrations below 1 g/l and sparging the fermentor with an O 2 -enriched air supply. Using the E. coli tryptophan promoter control of transcription, a 2.2 g/l production level of IL-1β was achieved in E. coli B at cell densities of 55 g dry weight per litre. Another genetic construction involving the bacteriophage lambda cI ts -P R expression cassette allowed a similar IL-1β production level (1.9 g/l) in E. coli E103S, albeit at a lower cell density (30 g/l). A simplified procedure allowing the purification of fully active IL-1β is also presented.

Constrained analogs of KCVFM with improved inhibitory properties against farnesyl transferase

Abstract Constrained analogs of KCVFM, reported thus far as one of the most active peptidic inhibitors of farnesyl transferase, have been synthesized. Replacement of Val-Phe with Val-Tic and (N-Me)Val-Tic led to dramatically more active analogs possessing favored extended conformations. Based on molecular modelling studies the design and synthesis of various conformational probes to be substituted for Val and Phe led to a good correlation between the ratio of extended conformers and biological activity.

Primary structure control of recombinant proteins using high-performance liquid chromatography, mass spectrometry and microsequencing.

The conformity of two recombinant proteins (a von Willbrand factor fragment and human serum albumin, consisting of respectively 289 and 585 amino acids) has been examined by HPLC combined with mass spectrometry and microsequencing, on both intact material and fragment peptides obtained by proteolytic cleavage. These studies confirmed that the primary structure of the recombinant proteins corresponds to that predicted from their gene, particularly the integrity of their N and C termini, and, in the case of albumin, the agreement between the observed disulfide bond pattern and the published model. Furthermore, the structure of an albumin-related compound could be elucidated. Application of LC-MS for batch-to-batch quality control is also under discussion.

Local constrained shifty pseudopeptides inhibitors of rasfarnesyl transferase

Abstract Pseudopeptide analogues related to the C-terminal tetrapeptide of ras-protein (Cys-Val-X-Met) were synthesized and evaluated for inhibition of ras farnesyl transferase (FTase). We demonstrate that the introduction of a shifty amino acid related to Cys instead of Cys-Val and a tetrahydroisoquinoline carboxylic acid (TIC) instead of Phe lead to potent inhibitors of FTase on isolated enzyme or on cell based tests. One of the pseudopeptides, conceived as a prodrug, suppressed specifically the ability of ras transformed cells to form colonies in soft agar.