Inefficient processing of human protein C in the mouse mammary gland

Abstract

Vitamin K-dependent plasma protein, human Protein C (HPC) has been expressed in transgenic mice, using a 4.2 kb mouse whey acidic protein (WAP) promoter, 9.0 kb HPC gene and 0.4 kb 3' flanking sequences. Expression was mammary gland-specific and the recombinant human Protein C (rHPC) was detected in milk at concentrations of 0.1 to 0.7 mg ml-1. SDS-PAGE revealed that the single, heavy and light chains of rHPC migrated with increased electrophoretic mobility, as compared to HPC. Enzymatic deglycosylation showed that these molecular weight disparities are in part due to differential glycosylation. The substantial increase observed in the amount of single chain protein, as well as the presence of the propeptide attached to 20-30% of rHPC, suggest that mouse mammary epithelial cells are not capable of efficient proteolytic processing of rHPC. The Km of purified rHPC for the S-2366 synthetic substrate was similar to that of plasma-derived HPC, while the specific activity was about 42-77%. Amino acid sequence analyses and low anticoagulant activity of purified rHPC suggest that gamma-carboxylation of rHPC is insufficient. These results show that proteolytic processing and gamma-carboxylation can be limiting events in the overexpression of fully biologically active rHPC in the mouse mammary gland.

Secretion of Human Furin into Mouse Milk

“…Transgene Construction and Generation of Transgenic Animals-The WAP/PACE construct comprising the 2.47-kb human furin cDNA and 74-base pair 3Ј-untranslated sequences, the four lines of CD-1 mice transgenic for WAP/HPC (12) and WAP/PACE expressing both transgenes in the mammary gland have been described (13).…”

“…The activity of furin varied in four lines of mice correlating with mRNA (13) and protein levels (Table I and In HPC/PACE bigenic mice, rHPC was processed to the mature protein (13), unlike rHPC from HPC transgenic mice, which consisted mainly of the single chain precursor with propeptide (12). Incubating the milk of bigenic and HPC mice together resulted in a reduction of the amount of rHPC single chain (Fig.…”

“…Transgenic animals secreting recombinant proteins into milk, blood, urine, or saliva (11) have been generated and differences in recombinant protein modification compared with the human counterpart observed (12). In a pioneering attempt to engineer the post-translational capacity of the mouse mammary gland, we reported enhanced proteolytic maturation of the recombinant human Protein C (rHPC) precursor upon coexpression with furin (13).…”

Modelos para a produção de eritropoietina recombinante humana in vivo e in vitro com vetores plasmideais em ovinos

“…As tentativas para a expressão de EPOrh no leite de animais transgênicos (ratos e coelhos) falharam em produzir uma proteína com pureza suficiente para condução dos estudos de glicosilação (DROHAN et al, 1994;SIMONS;CLARCK,, 1998;JAE et al, 2009e CASTRO et al, 2010. Outro Revisão de Literatura problema relacionado à produção de animais transgênicos para a expressão de EPOrh foi relatado por Sánchez et al (2004).…”

“…A mesma problemática foi encontrada no desenvolvimento de outros animais transgênicos que propunham a expressão da EPOrh no leite de ratos e coelhos (DROHAN et al, 1994;SIMONS;CLARCK, 1998;e JAE et al, 2009) e também na sua glicosilação incompleta (AGUIRRE; CASTRO-PALOMIND; DE LA FUENTE , 1998). Foi sugerido que a glândula mamária tem capacidade limitada para a glicosilação protéica que é reduzida quando é superior a 1mg/ml (RODRÍGUEZ; ARNAUD; BLANCHT, 1995 gelado (-20ºC), que foi armazenado no freezer -80ºC por no mínimo 48h.…”