title: Establishment and optimization of a wheat germ cell-free protein synthesis system and its application in venom kallikrein.
abstract: Wheat germ cell-free protein synthesis systems have the potential to synthesize functional proteins safely and with high accuracy, but the poor energy supply and the instability of mRNA templates reduce the productivity of this system, which restricts its applications. In this report, phosphocreatine and pyruvate were added to the system to supply ATP as a secondary energy source. After comparing the protein yield, we found that phosphocreatine is more suitable for use in the wheat germ cell-free protein synthesis system. To stabilize the mRNA template, the plasmid vector, SP6 RNA polymerase, and Cu(2+) were optimized, and a wheat germ cell-free protein synthesis system with high yield and speed was established. When plasmid vector (30 ng/μl), SP6 RNA polymerase (15 U), phosphocreatine (25 mM), and Cu(2+) (5 mM) were added to the system and incubated at 26°C for 16 h, the yield of venom kallikrein increased from 0.13 to 0.74 mg/ml. The specific activity of the recombinant protein was 1.3 U/mg, which is only slightly lower than the crude venom kallikrein (1.74 U/mg) due to the lack of the sugar chain. In this study, the yield of venom kallikrein was improved by optimizing the system, and a good foundation has been laid for industrial applications and for further studies.
authors.affiliation: School of Chemical Engineering and Technology, Tianjin University Tianjin 300072, China.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Wang
authors.forename: Wentao
authors.initials: W
authors.lastname: Xu
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Kou
authors.forename: Yunbo
authors.initials: Y
authors.lastname: Luo
authors.forename: Yanan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Biao
authors.initials: B
authors.lastname: Ma
authors.forename: Mengsha
authors.initials: M
authors.lastname: Wang
authors.forename: Kunlun
authors.initials: K
authors.lastname: Huang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001709:Biotechnology; D002474:Cell-Free System / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D003300:Copper / Q000378:metabolism; D007610:Kallikreins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014908:Triticum / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D014688:Venoms / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014688:Venoms; D003300:Copper; D000255:Adenosine Triphosphate; D007610:Kallikreins