Cloning, high expression and purification of recombinant human intereferon-beta-1b in Escherichia coli.
Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-beta-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 microg/ml has increased the specific product yield of 66 mg optical density(-1) l(-1) (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-beta-1b obtained in this study is the highest that has been reported so far.
Rao
Dasari Venkata Krishna
DV
Research and Development, Biotechnology Division, Natco Research Centre, Hyderabad, India. krishnaraodv@yahoo.co.in
Ramu
Chatadi Tulasi
CT
Rao
Joginapally Venkateswara
JV
Narasu
Mangamoori Lakshmi
ML
Rao
Adibhatla Kali Satya Bhujanga
AK
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011312:Pressure; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012293:Rifampin / Q000494:pharmacology; D012995:Solubility
D003470:Culture Media; D011994:Recombinant Proteins; D000068576:Interferon beta-1b; D007544:Isopropyl Thiogalactoside; D016899:Interferon-beta; D005947:Glucose; D012293:Rifampin