Accurately cleavable goat β-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland

Abstract

Poor expression is the key factor hampering the large-scale application of transgenic animal mammary gland bioreactors. A very different approach would be to evaluate the secretion of recombinant proteins into milk in response to a cleavable signal peptide of highly secreted lactoproteins. We previously reported rabbits harboring mammary gland-specific expression vector containing a fusion cDNA (goat β-lactoglobulin (BLG) signal peptide and recombinant human plasminogen activator (rhPA) coding sequences) expressed rhPA in the milk, but we did not realize the signal peptide contributed to the high rhPA concentration and did not mention it at that time. And the molecular structure and biological characteristics still remain unknown. So, rhPA in the milk was purified and characterized in the present study. rhPA was purified from the milk, and the purity of the recovered product was 98% with no loss of biological activity. Analysis of the N-terminal sequence, C-terminal sequence, and the molecular mass of purified rhPA revealed that they matched the theoretical design requirements. The active systemic anaphylaxis (ASA) reactions of the purified rhPA were negative. Taken together, these results indicated that the goat BLG signal peptide can efficiently mediate rhPA secretion into milk and was accurately cleaved off from rhPA by endogenous rabbit signal peptidase. We have reinforced the importance of a rhPA coding region fused to a cleavable heterologous signal peptide from highly secreted goat BLG to improve recombinant protein expression. It is anticipated that these findings will be widely applied to high-yield production of medically important recombinant proteins.

The High-Level Expression Analysis of RhPA In The RhPA/gGH Double-Transgenic Rabbits And Its Thrombolysis Activity In Vitro

“…Moreover, the strong thrombolytic activity of rhPA expressed in their whey was demonstrated by thrombolytic activity assay in vitro. The result demonstrated that the introduction of gGH gene can greatly promote the expression level of rhPA gene in the mammary gland of transgenic rabbits, and the expression level and thrombolytic activity in our study was significantly better [13][14][15][16][17][26][27] .…”

“…Therefore, it is highly possible to improve the expression level of exogenous genes in transgenic animal mammary gland by using GH gene introduction. However, the current strategy to improve the expression level of tPA and rhPA genes in the mammary gland of transgenic animals is often to optimize the gene vector construction [15][16][26][27] . There are few reports on the synergistic promotion of tPA gene expression by double transgenic animals, especially the study of GH gene synergistically promoting the expression of tPA in transgenic animals at home and abroad.…”

“…They were mammalian mammary gland-specific expression vectors with goat β-casein as regulatory element and CMV as promoter (Fig. 1), which have been validated for expression recombinant protein on goat cells, mice, and individual rabbits [16,[26][27] . FSH (Ningbo Sansheng Pharmaceutical Co. Ltd.), HCG (Lizhu Pharmaceutical Co. Ltd.), Sumianxin II(Veterinary Institute of Military University), FBS(HyClone), hyaluronidase(Sigma), Zoletil50(Virbac), protease K(Sigma), M2(Sigma), M16 (Sigma), mouse anti-tPA monoclonal antibody (Santa Cruz), goat anti-mouse monoclonal antibody IgG-HRP(Santa Cruz); DNA gel purification and recovery kit was purchased from QIAGEN, various restriction enzymes and DNA polymerases were purchased from Takara Bio (Dalian) Co. Ltd., other reagents were purchased from Shanghai Pharmaceuticals, Shanghai Shenggong Bioengineering Co. Ltd., Nanjing Shengxing Biotechnology Co. Ltd.…”

Evaluation of the α-casein (CSN1S1) locus as a potential target for a site-specific transgene integration

“…We made a full size replacement of the Csn1s1 ORF from start to stop codon and, as a consequence, the N-terminal Csn1s1 signal peptide (1–15 aa) was replaced with the N-terminal hGMCSF secretion signal (1–17 aa). It is known that native signal peptide could not compete in efficiency with the milk-specific secretion signals, such as BLG N-terminal fragment (1–18 aa) which could improve recombinant protein production by several orders 30 . We have tried to generate hGMCSF transgenic mouse line with preserved Csn1s1 signal peptide through pronuclear microinjection, using the same CRIPSR/Cas9 approach (donor vector + gRNA + Cas9) we conducted in ES cells (Fig.…”

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

“…This strategy is frequently employed to create "enriched" milk with improved composition (An et al, 2012;Wan et al, 2012;Yuan et al, 2014), or to achieve large scale production of recombinant human proteins in mouse models (Wu et al, 2012;Burkov et al, 2013;Qian et al, 2014) and in livestock species (Kalds et al, 2019). Milk-specific signal peptides are used in biotechnology to enhance recombinant protein secretion during lactation (Yu et al, 2006;Liu et al, 2014;Lu et al, 2019).…”