High-efficiency secretory expression and characterization of the recombinant type III human-like collagen in Pichia pastoris

Abstract

Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L^-1 was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation and showed the characteristics of Gly-X-Y repeated short peptide sequence. In amino acid analysis, glycine shared 27.02% and proline 23.92%, which were in accordance with the characteristics of collagen. Ultraviolet spectrum combined with Fourier transform infrared spectroscopy as well as mass spectrometry demonstrated that the target product conformed to the characteristics of collagen spectrums and existed as homologous dimer and trimer in the broth. This work provided a sustainable and economically viable source of the recombinant type III human-like collagen.

Keywords: Characterization; Fermentation; Human-like collagen; Protein purification; Secretory expression.

Fig. 1

A The construction process of hlCOLIII high-yielding strain. The target gene after hydrophobic amino acid substitution was inserted into the pPIC9K expression vector and introduced into GS115. The transformants grown on the MD screening plate were subjected to screening for multicopy strains on plates containing high concentrations of G418. B SDS-PAGE analysis of 9 recombinant strains selected from the plate containing high concentration of G418 (5 mg mL⁻¹). Lanes 1–9 were the expression levels of recombinant strains 1^#–9^# (loading volume 20 μL, 3 times concentrated by trichloroacetic acid), respectively, as well as lanes M₁ and M₂ were standard molecular weights. C Western blotting analysis of the expression products of 2^# recombinant strain. Lane 1 was the blank control group (GS115/pPIC9K), lane 2 was the experimental group (2^# GS115/pPIC9K-col), and lane M was standard molecular weights. D The expression levels of hlCOLIII were driven by 7 different promoters in cooperation with P_AOX1. Taking the recombinant strain GS115/pPIC9K-col (2^#) as the starting strain (control)

Fig. 2

A High-density fermentation culture of recombinant strains was carried out in a 5-L bioreactor. Glycerol feeding for some 4 h after 24th hour of inoculation and methanol was fed at 30th h after 2 h of starvation treatment. B SDS-PAGE analysis of broth supernatant after 36 h of inoculation. Lanes 1–9 were cultured 36, 42, 48, 54, 60, 66, 72, 78, and 84 h (loading volume 20 μL, not concentrated), respectively, lanes M1 and M2 were protein ladder. C Affinity purification of expression products. D The purified product exhibited a single band verified by SDS-PAGE. Lane 1 was supernatant of broth before purification, lane 2 was purified product, and lane M was standard molecular weights

Fig. 3

A The protein sequences of N-terminal and C-terminal of the expressed product. B The ultraviolet full-scan spectrum of hlCOLIII in the range of 200–400 nm. The absorption peaks occur at 223.2 and 278.8, respectively, due to the electronic transitions of the different groups

Fig. 4

A MALDI-TOF-MS analysis of hlCOLIII shows that there are three peaks with decreasing intensity appeared at 11313.4, 22643.0 and 33901.2, respectively, and the m/z values of three were in an integral multiple relationship. B Native PAGE analysis of hlCOLIII. Lanes 1–3: hlCOLIII samples, with one band at 55 kDa and one between 70 and 100 kDa, respectively; Lane M: protein ladder

Fig. 5

FTIR spectra of hlCOLIII displaying absorption peaks of amide A, amide B, and amide I–III