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Studies of the scale-up of production and recovery of recombinant proteins formed as inclusion bodies.

Jin, Kai; (1992) Studies of the scale-up of production and recovery of recombinant proteins formed as inclusion bodies. Doctoral thesis (Ph.D.), University College London. Green open access

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Abstract

This is a study of the production of two recombinant proteins, prochymosin and a miniantibody (formed as inclusion bodies) and the subsequent recovery of the former at pilot plant scale. Recombinant E.coli strains which contain temperature-sensitive multicopy plasmids carrying foreign genes have been employed in this study. The scale-up work involved fermentations, up to 100 litre in capacity, and recovery of expressed prochymosin inclusion bodies. A number of different scales of fermentations (20L, 42L & 100L) were examined to determine the fermentation process suitable for large-scale production of recombinant prochymosin in E.coli HB101 pMG168. Fermentations at 20L scale were also performed for expression of recombinant mini-antibody in E.coli CAG629 pMG LF9. Fermentation media were defined to allow scale-up of production of recombinant prochymosin: a cheap nutrient source was evolved and antibiotic was removed, allowing low costs and facilitating safety while retaining plasmid stability. Thermal induction of cells at different stages during growth was examined. Expression of recombinant prochymosin was only achieved when cells were induced during the exponential growth phase. Furthermore, higher yields of prochymosin were obtained by inducing cells at later stages in the growth phase. An industrial high pressure homogeniser was employed to examine the disruption of recombinant E.coli cells. Repeated homogenisation resulted in the reduction of cell debris size without significantly affecting inclusion body particle size, thus easing subsequent separation of the inclusion body fraction from cell debris by centrifugation. The separation of prochymosin inclusion bodies from cell debris was studied in an industrial disc stack centrifuge. Deviations were found between real performance of an industrial disc centrifuge and theoretically predicted performance. A method has been developed to monitor disc centrifuge performance by turbidimetric ratio measurements at two wavelengths, and this method has potential application in developing an automatic controlling system for optimal recovery of inclusion bodies. A variety of techniques for protein analysis have been applied to investigations on the productivity of expression of recombinant prochymosin from fermentations, on the disruption of recombinant cells in an industrial high pressure homogeniser and on the separation of prochymosin inclusion bodies from cell debris in an industrial disc stack centrifuge. These techniques include high performance liquid chromatography (HPLC), particle analysis and protein gel electrophoretic techniques. In addition, various gel elution methods for purification and direct quantitation of protein species, in addition to scanning densitometry, have been employed.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Studies of the scale-up of production and recovery of recombinant proteins formed as inclusion bodies.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
URI: https://discovery.ucl.ac.uk/id/eprint/10109265
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