Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

Abstract:

:Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Horstick EJ,Jordan DC,Bergeron SA,Tabor KM,Serpe M,Feldman B,Burgess HA

doi

10.1093/nar/gkv035

subject

Has Abstract

pub_date

2015-04-20 00:00:00

pages

e48

issue

7

eissn

0305-1048

issn

1362-4962

pii

gkv035

journal_volume

43

pub_type

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