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.
Research and Development, Biotechnology Division, Natco Research Centre, Hyderabad, 500018, India. nrc@natcopharma.co.in
Dasari V
DV
Krishna Rao
Joginapally V
JV
Rao
Mangamoori L
ML
Narasu
Adibhatla Kali S
AK
Bhujanga Rao
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