title: Screening and identification of genetic loci involved in producing more/denser inclusion bodies in Escherichia coli.
abstract: BACKGROUND
  Many proteins and peptides have been used in therapeutic or industrial applications. They are often produced in microbial production hosts by fermentation. Robust protein production in the hosts and efficient downstream purification are two critical factors that could significantly reduce cost for microbial protein production by fermentation. Producing proteins/peptides as inclusion bodies in the hosts has the potential to achieve both high titers in fermentation and cost-effective downstream purification. Manipulation of the host cells such as overexpression/deletion of certain genes could lead to producing more and/or denser inclusion bodies. However, there are limited screening methods to help to identify beneficial genetic changes rendering more protein production and/or denser inclusion bodies.
  
  
  RESULTS
  We report development and optimization of a simple density gradient method that can be used for distinguishing and sorting E. coli cells with different buoyant densities. We demonstrate utilization of the method to screen genetic libraries to identify a) expression of glyQS loci on plasmid that increased expression of a peptide of interest as well as the buoyant density of inclusion body producing E. coli cells; and b) deletion of a host gltA gene that increased the buoyant density of the inclusion body produced in the E. coli cells.
  
  
  CONCLUSIONS
  A novel density gradient sorting method was developed to screen genetic libraries. Beneficial host genetic changes could be exploited to improve recombinant protein expression as well as downstream protein purification.
authors: BioChemical Sciences & Engineering, E.I DuPont India Pvt. Ltd. DuPont Knowledge Centre, Survey No 542/2. DS-9, ICICI Knowledge Park, Ranga Reddy District, Turkapally, Hyderabad 500078, India.
authors: Neeraj
authors: N
authors: Pandey
authors: Annapurna
authors: A
authors: Sachan
authors: Qi
authors: Q
authors: Chen
authors: Kristin
authors: K
authors: Ruebling-Jass
authors: Ritu
authors: R
authors: Bhalla
authors: Kiran Kumar
authors: KK
authors: Panguluri
authors: Pierre E
authors: PE
authors: Rouviere
authors: Qiong
authors: Q
authors: Cheng
mesh_terms: D002499:Centrifugation, Density Gradient; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D055786:Gene Knockout Techniques; D015723:Gene Library; D056426:Genetic Loci; D002479:Inclusion Bodies / Q000378:metabolism*; D011205:Povidone / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone
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