Enhancement of recombinant human ADAM15 disintegrin domain expression level by releasing the rare codons and amino acids restriction.
This study was aimed at increasing the production of the recombinant human ADAM15 disintegrin domain (RADD) in Escherichia coli by releasing the rare codons and restricting amino acid residues. Three different strategies for increasing RADD production were examined: to select the suitable host strain, to optimize the rare codons, and to delete the amino acids residues. The total fusion protein glutathione-S-transferase (GST)-RADD concentration of 298 mg/l and 326 mg/l were achieved by selecting E. coli Rosetta (DE3) as the host strain and by changing GGA to GGC at the GST-RADD cassette, respectively. The RADD concentration was increased by 35.7% by eliminating the "Pro-Glu-Phe" residues at the GST-RADD junction. By combinational utilizing the preferred codon introduction and amino acid sequence optimization in E. coli Rosetta (DE3), the highest RADD concentration of 68 mg/l was achieved. The proposed strategy may provide an alternative approach for other enhanced recombinant protein production by E. coli.
Wu
Jing
J
Department of Pharmaceutical and Molecular Biotechnology, School of Medicine & Pharmaceutics, Jiangnan University, Wuxi, Jiangsu 214122, People's Republic of China.
Zhang
Lianfen
L
Lei
Jianyong
J
Cai
Gangming
G
Zhu
Wei
W
Lu
Daru
D
Jin
Jian
J
D051722:ADAM Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D003062:Codon / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D015203:Reproducibility of Results; D013917:Thrombin / Q000378:metabolism
D000596:Amino Acids; D003062:Codon; D008565:Membrane Proteins; D011994:Recombinant Proteins; D013917:Thrombin; D051722:ADAM Proteins; C098660:ADAM15 protein, human