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.

Keywords: BLG; mammary gland bioreactor; purification; rabbit; rhPA; signal peptide.

Figure 1. Theoretical amino acid sequence of the cDNA

The amino acid sequence of the goat β-casein signal peptide is shown in orange, the amino acid sequence of the K2 domain of recombinant human plasminogen activator is in blue, the amino acid sequence of the P domain of recombinant human plasminogen activator is in green.

Figure 2. Identification of protein fractions eluted from various chromatography columns

(A) Identification of purified rhPA by SDS-PAGE. 1: Filtrate from the 100 kDa ultrafilter; 2: eluate of benzamidine affinity chromatography; 3: eluate of cation exchange chromatography; 4: eluate of cibacron blue affinity chromatography; 5: commercially available Alteplase (Actilyse, Boehringer Ingelheim International GmbH, Germany); M: RealBand Pink Blue Protein Marker (Sangon Biotech, China). (B) Identification of purified rhPA by Western blot. 1: Commercially available Alteplase, two-chain form; 2: purified rhPA.

Figure 3. Identification of fibrinolytic activity of the purified rhPA

Wells were filled with 20 μl aliquots of samples. Wells 1–7: Alteplase 1000, 500, 250, 125, 62.5, 31.25, and 0 ng/μl with circle diameters of 20, 18, 16, 14, 9, 8, and 6 mm, respectively; 8: null; 9, 10: milk samples of wild-type rabbits diluted 40 times with Buffer A; 11: 3 ng/μl of purified rhPA, circle diameter = 16 mm; 12: milk sample of transgenic rabbits diluted 40 times with Buffer A, circle diameter = 16 mm.

Figure 4. Circular dichroism spectra of Alteplase and rhPA

Proteins (0.1 mg/ml) in 50 mM phosphate buffer (pH 7.4) was measured with a 1 mm cell in the far-UV region.

Figure 5. Changes in IgE and histamine in each group of guinea pigs after stimulation

(A) Changes of IgE in guinea pigs after stimulation. (B) Changes of histamine in guinea pigs after stimulation. N=7 for solvent (0.225 mol/L l-arginine, 0.1114 mol/L phosphoric acid, 0.012% w/v polysorbate 80, pH 7.2), BSA (10 mg/ml bovine serum albumin, A1933, Sigma–Aldrich, Germany + solvent), Alteplase (1.12 mg/ml Alteplase + solvent), and rhPA (1.12 mg/ml rhPA + solvent). Data are expressed as the mean ± S.E.M. P-values were determined by one-way ANOVA followed by a post-hoc Dunnett’s test. **P<0.01 vs negative group.