Fig 4 - uploaded by Yiqiang Zhao
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The tertiary structures of the main domains of casein proteins, 10 recombinant human proteins highly expressed in the milk of transgenic animal, and two moderately expressed proteins CEL (1.00 mg/mL) and Calc1(2.10 mg/mL) based on cDNA expression constructs. Figures were made using VMD (http://www.ks.uiuc.edu/ Research/vmd/) and rendered using Snapshot
Source publication
Animal mammary glands have been successfully employed to produce therapeutic recombinant human proteins. However, considerable variation in animal mammary transgene expression efficiency has been reported. We now consider whether aspects of codon usage and/or protein tertiary structure underlie this variation in mammary transgene expression.
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Citations
... The full-length hFVIII cDNA contains an open reading frame encoding a polypeptide of 2351 amino acids [4]. This primary translation product contains a 19-amino-acid signal peptide and six homologous domains in the order A1-A2-B-A3-C1-C2 [5]. The full-length hFVIII cDNA exceeds the compact volume of most vectors [6]. ...
... After the recombinant protein is secreted into milk, a pharmaceutical product could be readily isolated and purified [14]. Therefore, milk is a better candidate source for mass production of functional proteins [5]. ...
Hemophilia A is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. The conventional treatment involves the administration of recombinant human FVIII (rhFVIII) preparations. In this study, the mammary gland 'bioreactor' is designed to specifically and efficiently express a foreign protein hFVIII in the mammary glands of transgenic mice. We constructed a P1A3-hFVIIIBD vector directed by the mammary gland-specific P1A3 promoter, and transiently transfected HC11 cells and mouse mammary glands with P1A3-hFVIIIBD or CMV-hFVIIIBD vectors directed by a ubiquitous cytomegalovirus (CMV) promoter, respectively. We also generated P1A3-hFVIIIBD and CMV-hFVIIIBD transgenic mice by microinjection, respectively. Our data indicated that both vectors effectively expressed hFVIIIBD in HC11 cells at the transcription level, and hFVIIIBD protein was efficiently expressed in mouse milk after the injection of the hFVIIIBD vectors into mouse mammary glands during lactation. In both CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice, hFVIIIBD proteins were efficiently expressed in the mammary glands at the mRNA and protein levels. No significant difference was observed in hFVIIIBD levels between the CMV-hFVIIIBD and P1A3-hFVIIIBD transgenic mice (P > 0.05). However, the activity of hFVIII in CMV-directed transgenic mice was slightly higher than that in P1A3-directed transgenic mice (P < 0.05). While hFVIIIBD was present in multiple organs in CMV-hFVIIIBD mice, P1A3-hFVIIIBD mice showed negligible hFVIIIBD expression in organs other than the mammary glands. This study demonstrated that the mammary gland-specific P1A3-hFVIIIBD vector was more suitable for the generation of hFVIIIBD mammary gland bioreactor.
... After examining mRNA expression, we further evaluated GH protein contents with RIA and ELISA. In the control group, GH increased slowly over time in Bcap-37 cells, implicating different GH domains and 4 casein types (He et al., 2010). In the two treatment groups, GH showed dramatic changes. ...
The aim of this study was to construct a mammary gland-specific expression vector, pGN, and to validate its function in expressing growth hormone (GH) both in vitro and in vivo. First, the GH gene was amplified and inserted downstream of the b-casein 5'-arm. Next, the neo gene was cloned downstream of the b-casein 3'-arm as a selection marker. To analyze the bioactivity of the pGN plasmid, we expressed pGN in a Bcap-37 cell line and in the goat mammary gland. Quantitative PCR analysis revealed that the expression of GH mRNA in the pGN-transfected group was higher than that of the control group in Bcap-37 cells. Results of a radioimmunoassay and an enzyme-linked immunosorbent assay demonstrated that the pGN-transfected group expressed much more GH protein than the non-transfected group (P < 0.05). Upon injection of the pGN plasmid into the goat mammary gland, GH mRNA and growth hormone receptor mRNA expressions increased 2-fold. In vivo analyses revealed that GH protein expression was higher in the injected group than in the control group. Together, these results strongly demonstrated that the pGN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing GH both in vitro and in vivo.
