Incorporation of partial polyhedrin homology sequences (PPHS) enhances the production of cloned foreign genes in a baculovirus expression system.
Baculovirus expression vector systems (BEVSs) have been used extensively for high-level expression of cloned foreign genes. In many instances, the levels of recombinant protein(s) produced in insect cells and larvae are insufficient for experimental purposes. Thus new techniques and methods are needed to increase significantly the protein expression levels in BEVS. In the present paper, we describe the incorporation of a 15 bp element derived from the 5'-end partial sequence of the polyhedrin gene, which contains the non-coding sequence ATAAAT and the coding sequence ATGCCGAAT, into the 5'-end of the CTB (cholera toxin B subunit)-INS (insulin) fusion gene. With the addition of the PPHS (partial polyhedrin homology sequences), two extra amino acids (Pro-Asn) were added to the N-terminus of the mCTB-INS (modified CTB-INS) fusion protein. This new fusion protein was expressed in both insect cells and larvae using BEVSs. We found that the addition of PPHS enhanced 4-fold the expression of CTB-INS in both insect cells and larvae. Further analysis revealed that the additional two amino acids in mCTB-INS did not significantly affect binding affinity for G(M1) ganglioside. Therefore the PPHS can be used as a constitutive element immediately downstream of the polyhedrin promoter to induce significant increases in the expression levels of cloned foreign genes.
Gong
Zhaohui
Z
Institute of Biochemistry, College of Life Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, People's Republic of China.
Jin
Yongfeng
Y
Zhang
Yaozhou
Y
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
D004268:DNA-Binding Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C085605:polyhedrin promoter-binding protein, insect