Characterization of humanized antibodies secreted by Aspergillus niger.

Two different humanized immunoglobulin G1(kappa) antibodies and an Fab' fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex(6)GlcNAc(2) to Hex(15)GlcNAc(2). An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

Oligomerization of the E5 protein of human papillomavirus type 16 occurs through multiple hydrophobic regions.

The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein's ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5.