Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Biochemical study of sortase E2 from Streptomyces mobaraensis and determination of transglutaminase cross-linking sites.

Distinct streptomycetes such as Streptomyces mobaraensis produce the protein cross-linking enzyme transglutaminase. Bioinformatic analysis predicted the occurrence of seven sortases exerting transpeptidation reactions similarly to transglutaminase. Here, we report the production and characterization of sortase E2 (Sm-SrtE2) solubilized by removal of its membrane anchor domain. Sm-SrtE2 activity was measured using pentapeptides predicted to be cell wall sorting signals of putative sortase substrate proteins. Preferred linkage to Gly3 by Sm-SrtE2 was in the order LAETG>>LAHTG>>LAQTG~LANTG>LARTG. Chaplin 1 from S. mobaraensis was further demonstrated to be an excellent substrate of both the intrinsic Sm-SrtE2 and transglutaminase. The unexpected discovery showing Gln-62 and Gln-65 of Δ1-50 -Sm-SrtE2 as transglutaminase cross-linking sites suggests that low enzyme stability might be due to anchor domain truncation and a disordered N terminus.