Expression, characterization, and application of human-like recombinant gelatin

Abstract

Gelatin is a product obtained through partial hydrolysis and thermal denaturation of collagen, belonging to natural biopeptides. With irreplaceable biological functions in the field of biomedical science and tissue engineering, it has been widely applied. The amino acid sequence of recombinant human-like gelatin was constructed through a newly designed hexamer composed of six protein monomer sequences in series, with the minimum repeating unit being the characteristic Gly-X-Y sequence found in type III human collagen α1 chain. The nucleotide sequence was subsequently inserted into the genome of Pichia pastoris to enable soluble secretion expression of recombinant gelatin. At the shake flask fermentation level, the yield of recombinant gelatin is up to 0.057 g/L, and its purity can rise up to 95% through affinity purification. It was confirmed in the molecular weight determination and amino acid analysis that the amino acid composition of the obtained recombinant gelatin is identical to that of the theoretically designed. Furthermore, scanning electron microscopy revealed that the freeze-dried recombinant gelatin hydrogel exhibited a porous structure. After culturing cells continuously within these gelatin microspheres for two days followed by fluorescence staining and observation through confocal laser scanning microscopy, it was observed that cells clustered together within the gelatin matrix, exhibiting three-dimensional growth characteristics while maintaining good viability. This research presents promising prospects for developing recombinant gelatin as a biomedical material.

Keywords: Pichia pastoris; Biomedical materials; Characterization; Monomer protein sequence; Recombinant human gelatin; Three- dimensional culture.

Fig. 1

A Sketch map of construction of recombinant P. pastoris GS115 with hlrGEL6 gene(gel6). The circle graph: Schematic map of the recombinant expression vector pPICZα-gel6. The gel6 gene was cloned into pPICZa-B using XhoI and NotI sites as described in Materials and methods

Fig. 2

Construction and transformation of recombinant vectors. (a) Identification of pPICZα-gel6 recombinant vector by double digestion, M: DNA marker, 1: Recombinant, 2: empty vector. 3. Double enzyme digestion vector. (b) Linearization of pPICZα-gel6, M: DNA marker, 1: before linearization, 2: after linearization. (c) Electrophoretic detection of PCR products of transforming strain, M: DNA marker, 1–20: PCR identification of different clones, - : negative control (empty vector), +: positive control (recombinant vector)

Fig. 3

SDS–PAGE and Western blot analysis of hlrGEL6 prepared by small shake tubes (a) SDS-PAGE was used to detect the culture supernatant. (b) Western blot detection of the culture supernatant(Anti-His-HRP). M: Protein marker, 1–10: positive colonies, -:Negative control (non-reductive)

Fig. 4

Analysis of the purification results of hlrGEL6 prepared by shake flask fermentation (a) The purified protein was detected by SDS-PAGE. (b) The protein secretion was detected by Western blot. M: protein marker, (1) Intracellular sample after induction, (2) Fermentation supernatant sample, (3) effluent sample, (4) cleaning fluid sample, (5) elution sample

Fig. 5

Molecular weight of hlrGEL6

Fig. 6

SEM analysis of hlrGEL6 (a) The porous structure of hlrGEL6. (b) Diameter of the hlrGEL6. (c) The porous structure of commercially available gelatin. (d) The diameter of commercially available gelatin

Fig. 7

Three-dimensional cell culture of hlrGEL6 hydrogel (a-d) hlrGEL6-HepG2 group. (a) The cells cultured in hlrGEL6 hydrogel appear rounded and form clusters. (b-c) The are one of the cell aggregates that are formed in hlrGEL6 hydrogel culture. (d) This is a screenshot of video images displayed HepG2 cells cultured in hlrGEL6. (e) gelatin-HepG2 group. (f) HepG2 group