Optimization of the AT-content of codons immediately downstream of the initiation codon and evaluation of culture conditions for high-level expression of recombinant human G-CSF in Escherichia coli.
Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl beta-D: -1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD(600) value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 microg/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.
Krishna Rao
Dasari V
DV
Research and Development, Biotechnology Division, Natco Research Centre, Hyderabad, 500018, India. nrc@natcopharma.co.in
Rao
Joginapally V
JV
Narasu
Mangamoori L
ML
Bhujanga Rao
Adibhatla Kali S
AK
D001482:Base Composition; D003062:Codon / Q000235:genetics*; D018387:Codon, Initiator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis / Q000187:drug effects; D011994:Recombinant Proteins; D012293:Rifampin / Q000494:pharmacology
D003062:Codon; D018387:Codon, Initiator; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D012293:Rifampin