Construction Of The Human Zona Pellucida Protein 3 Mutant And Its Expression In Pichia Pastoris

In the present paper, site-directed mutagenesis was used to convert three residues in the amino acid sequence of human zona pellucida protein 3 (hZP3) that were supposed to be the potential cleavage sites which may be digested by the proteases in the Pichia pastoris. The intact recombinant human zona pellucida protein 3(rhZP3) was expected to be obtained by removing these protease cleavage sites. In addition, the other aim of our work is to investigate the influence of C-terminal transmembrane-like domain and polyhistidine tag position on the expression and purification of rhZP3 produced in Pichia pastoris. C-terminal transmembrane-like domain of hZP3, although removed as the proteins proceed through the secretory pathway and not present in the mature proteins, possibly has the effects on the protein expression due to its hydrophobic group. Also, the polyhistidine tag position in the subcloned gene enables to affect the protein expression and purification. For these purposes, experiments were designed as follows: pPIC9K-6×his and pPICZaA were used as the expression vectors. Specifically, pPIC9K-6×his was modified by inserting a DNA fragment coding 6-histidine and 2-glycin in the multiple cloning site of pPIC9K for expressing the fusion protein with a N-terminal polyhistidine tag. Then four mutated hZP3 gene fragments were produced by overlap extension PCR according to the sequences with or without C-terminal transmembrane-like domain and the restriction digestion sites which will be introduced to them. After enzyme digestion and ligation, they were successfully cloned into pPIC9K-6xhis and pPICZaA, respectively. Have been linearized with SacI, four recombinant expression vectors were all transformed into GS115 to express the rhZP3 proteins. The results of our investigations indicated that: (1) The degradation of rhZP3 was successfully inhibited by point mutations. (2) C-terminal transmembrane-like domain and polyhistidine tag position had few effects on the expression of the rhZP3. (3) polyhistidine tag position did not greatly affect the purification behavior.