A high cell density fermentation strategy to produce recombinant malarial antigen in E. coli.
A high cell density cultivation method was developed to produce recombinant PvRII, a malaria vaccine candidate, in E. coli for use in vaccine studies. Cells were grown in completely defined media and glucose was fed to achieve a specific growth rate of 0.12 h(-1) until cells reached 55 g dry wt l(-1). Culture was then induced with 1 mM: IPTG and cells were further grown for 4 h to reach 85 g dry wt l(-1) at 0.1 h(-1). Recombinant PvRII was purified from inclusion bodies under denaturing conditions using metal affinity chromatography which yielded 10 mg PvRII g(-1) dry wt. After refolding, PvRII was greater than 98% pure, homogeneous and functionally active in that it specifically bound Duffy positive human red cells.
Yazdani
Syed Shams
SS
Malaria Research Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India. shams@icgeb.res.in
Shakri
Ahmad Rushdi
AR
Chitnis
Chetan E
CE
D000953:Antigens, Protozoan / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005285:Fermentation / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D015800:Protozoan Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis
D000953:Antigens, Protozoan; C066001:Duffy antigen binding protein, Plasmodium; D015800:Protozoan Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins