Advertisement

Microbial Cell Factories

, 5:P54 | Cite as

Increasing the quality of recombinant products – Higher attraction of ribosomes leads to suppression of secondary ribosome binding sites

  • Ulf Liebal
  • Olli Niemitalo
  • Anu Mursula
  • André Juffer
  • Peter Neubauer
Open Access
Poster Presentation
  • 1k Downloads

Keywords

Start Codon Translation Initiation Initiation Factor Translation Ribosome Binding Site Wnt4 Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Background

In translation initiation the 3' end of the 16s rRNA binds to the complementary Shine Dalgarno (SD) sequence. Together with bound initiation factors translation can subsequently begin at the AUG start codon. However, there may be SD related sequences throughout the coding region of the mRNA. These secondary SD sequences can recruit ribosomes as well, in particular if they are embedded in purine rich regions [1]. The existence of such secondary ribosome binding sites can greatly reduce the expression efficiency since ribosomes recruited to the secondary SD site hinder elongating ribosomes in their progression. If a start codon is nearby a secondary SD site even truncated protein could build up in expense of full length protein [2].

Results

In our attempts to increase the production of recombinant Wnt4 protein in E. coli BL21(DE3) we optimised the 5' coding sequence by secondary structure modelling with silent mutations to promote the single stranded nature of the translation initiation region of the mRNA. Interestingly, a major result of this optimisation, which was performed to provide higher ribosome loading to the wnt mRNA, was the disappearance of a shorter variant of Wnt4, which was formed due a second internal ribosome binding site (nucleotides 90 to 97).

Conclusion

As no other properties of the expression system and conditions were changed we argue that a higher ribosome loading from the regular SD site raises the ribosome coverage of the mRNA such that the secondary ribosome binding site is obscured by translating ribosomes. As a result the production of truncated protein is reduced.

Notes

Acknowledgements

This study was supported by the TEKES "Neobio" programme and a grant to AM by the Academy of Finland.

References

  1. 1.
    Ivanov I, Alexandrova R, Dragulev B, Saraffova A, AbouHaidar MG: Effect of tandemly repeated AGG triplets on the translation of CAT-mRNA in E. coli. FEBS Lett. 1992, 307: 173-176. 10.1016/0014-5793(92)80761-5.CrossRefGoogle Scholar
  2. 2.
    Ozin AJ, Costa T, Henriques AO, Moran CP: Alternative translation initiation produces a short form of a spore coat proteinin Bacillus subtilis. J Bacteriol. 2001, 183: 2032-2040. 10.1128/JB.183.6.2032-2040.2001.CrossRefGoogle Scholar

Copyright information

© Liebal et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd.

Authors and Affiliations

  • Ulf Liebal
    • 1
  • Olli Niemitalo
    • 1
  • Anu Mursula
    • 1
  • André Juffer
    • 2
  • Peter Neubauer
    • 1
  1. 1.Bioprocess Engineering Laboratory, Dept. Process & Environm. Engin. and Biocenter OuluUniversity of OuluOuluFinland
  2. 2.Triacle BiocomputingOulu

Personalised recommendations