Expression of recombinant Hirudin in transgenic mice milk driven by the goat β‐casein promoter

Yen, Chon-Ho; Yang, Chi-Kai; Chen, I-Chung; Lin, Yin-Shen; Lin, Chih‐Sheng; Chu, Sen; Tu, Ching-Fu

doi:10.1002/biot.200800069

Cited by 9 publications

(1 citation statement)

References 42 publications

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“…The tyrosine O -sulfate in tgFIX was determined by DABS-Cl method as described previously [20]. Activation peptide of tgFIX was digested with trypsin and purified by reverse phase HPLC [21].…”

Section: Methodsmentioning

confidence: 99%

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Lee

Lin

et al. 2014

BioMed Research International

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Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

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Section: Methodsmentioning

confidence: 99%

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Lee

Lin

et al. 2014

BioMed Research International

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Recombinant Protein Expression in Milk of Livestock Species

Bösze

Hiripi

2011

Recombinant Gene Expression

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Producing complex recombinant proteins in the milk of transgenic animals offers several advantages: large amounts of proteins can be obtained, and in most cases, these proteins are properly folded, assembled, cleaved, and glycosylated. The level of expression of foreign genes in the mammalian gland cannot be predicted in all cases, and appropriate vectors must be used. The main elements of these vectors are as follows: a well-characterized specific promoter, the coding region of the gene of interest, preferably with a homologous or heterologous intron, to improve transcription efficiency, and an insulator or boundary element to counteract the chromosomal position effects at the integration site. Once high expression levels are achieved, and the recombinant protein is purified, an essential step in the analysis of the final product is determining its degree of glycosylation. This is an important readout because it can affect among other parameters the stability and immunogenicity of the recombinant protein.

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Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

Huang

Zhang

et al. 2012

Journal of Industrial Microbiology and Biotechnology

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Hirudin variant III (HV3) is potentially useful in the prevention and treatment of cataracts. To prepare sufficient amounts of rHV3 for further preclinical studies, we developed an effective process for robust preparative-scale extracellular production of rHV3 in Escherichia coli. In a 7-l bioreactor, under the optimal fed-batch fermentation conditions, rHV3 was excreted into the culture supernatant and yielded up to 915 mg l(-1). Then, a four-step purification procedure was applied to the product, which included ultrafiltration, hydrophobic chromatography, anion-exchange chromatography, and preparative reversed-phase fast protein liquid chromatography (FPLC). The overall maximum recovery attained was 56 %, the purity reached at least 99 % as evaluated by HPLC analysis, the molecular weight was determined to be 7,011.10 Da by matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and the pI was 4.46 as analyzed by isoelectric focusing. The N- and C-terminal sequence analysis confirmed the product homogeneity. The final product contained at most 10 pg of residual DNA per dose (0.2 mg) of rHV3 by high-sensitivity hybridization assay and at most 3 EU endotoxin protein/mg by limulus amebocyte lysate assay. Taken together, the rHV3 produced in multigram quantities in E. coli by this bioprocess meets the regulatory criteria for biopharmaceuticals and can be used as a drug candidate for preclinical studies.

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Expression of recombinant Hirudin in transgenic mice milk driven by the goat β‐casein promoter

Cited by 9 publications

References 42 publications

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Recombinant Protein Expression in Milk of Livestock Species

Robust preparative-scale extracellular production of hirudin in Escherichia coli and its purification and characterization

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