Expression, purification, and characterization of recombinant human consensus interferon-alpha in Escherichia coli under λP(L) promoter.
Recombinant human consensus interferon-alpha (cIFN-α) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-α gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45-50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-α gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-α (Power3-cIFNα) plasmid carried the cIFN-α gene under transcriptional regulation of the heat-inducible λP(L) promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-α protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-α protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-α anti-cancer activity was assayed and compared with the commercially available IFN-α 2a.
Antibody Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Alexandria, Egypt.
Y
Y
Mohammed
N A
NA
El-Baky
E M
EM
Redwan
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003062:Codon; D016384:Consensus Sequence; D003470:Culture Media; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006358:Hot Temperature; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D003062:Codon; D003470:Culture Media; D017931:DNA Primers; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins