Fusion expression vectors for recombinant gene products processed easily and purified rapidly by affinity chromatography.
A DNA fragment encoding IgG-binding domain B,C (PABC) was separated from protein A gene, cloned into phage M13 and modified by oligo-directed mutagenesis at the hydroxylamine-cleaved site from Asn-Gly to Asn-Ala in domain B and C, respectively. The modified PABCm gene fragment was used to construct one set of fusion expression vectors in different reading frames. Processing sequences such as those recognized by enterkinase, collagenase, thrombin, activated factor X and cleaved by the hydroxylamine, N-chlorosuccinimide etc. can be created in the fusion site. Using the above vectors, fusion proteins such as PABCm-IGF-I, -hGRF, -bGRF and their derivatives were highly expressed in E. coli. The yield of fusion proteins is over 100 mg per liter cultured by analysis of SDS-PAGE. The PABC fusion proteins can be rapidly purified by the affinity chromatography with a IgG-sepharose column.
Li
B
B
Shanghai Institute of Biochemistry, Chinese Academy of Sciences.
Yang
X
X
Jiang
Z
Z
Hu
M
M
Liang
Z
Z
D001483:Base Sequence; D002846:Chromatography, Affinity; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007334:Insulin-Like Growth Factor I / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D011401:Promoter Regions, Genetic; D016364:Reading Frames / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D007334:Insulin-Like Growth Factor I