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. 2008 May 14;3(5):e2189.
doi: 10.1371/journal.pone.0002189.

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Affiliations

Heterologous protein expression is enhanced by harmonizing the codon usage frequencies of the target gene with those of the expression host

Evelina Angov et al. PLoS One. .

Abstract

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm ("codon harmonization") for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the "recoded" genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.

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Conflict of interest statement

Competing Interests: Coauthors, EA, RLK and JAL are listed as co-inventors on a patent application for the algorithm, codon harmonization.

Figures

Figure 1
Figure 1. Comparison of Codon Usage Frequencies in the Native MSP142 (FVO) and Recoded Sequences as a Function of Expression Host, for either native P. falciparum or E. coli expression.
Amino acid segment of MSP142 (FVO) sequence is shown (residue # 140–159) with the codon usage frequencies rounded to the nearest 10%. The shaded residue at I(141) was targeted for synonymous replacement in FMP003.
Figure 2
Figure 2. Comparison of MSP142 (FVO) expression from plasmids encoding either wild type or the FMP003 gene by SDS-PAGE and Coomassie Blue staining or Western Blotting.
Panel A, Coomassie Blue stained gels at the time of induction (U) or 3 hours post induction (I 3hr); Panel B, Western blots at the time of induction (U) or 3 hours post induction (I 3hr); Panel C, Coomassie Blue stained gels of protein following affinity purification on Ni+2-NTA chromatography. Arrows indicate migration of FMP003.
Figure 3
Figure 3. Comparison of expression levels of (WT), FMP003, and FMP010 proteins.
Total E. coli cell lysates were separated by SDS-PAGE followed by staining with Coomassie Blue at either the time of induction (U) or 3 hours post induction (I 3hr) with 0.1 mM IPTG. The arrow indicates the migration of MSP142 protein.
Figure 4
Figure 4. Solubility of the FMP010 protein product.
U, lysate from uninduced cells; I3hr, lysate prepared 3 hours after induction with 0.1 mM IPTG; f1, 30,000×g supernate from induced cell lysate; f2, 30,000×g pellet from induced cell lysate; f3, 100,000×g supernate of 30,000×g supernate; f4, 100,000×g pellet of 30,000×g supernate,. The arrow indicates the migration of FMP010.
Figure 5
Figure 5. Comparison of expression levels of wild type (WT) and full gene codon harmonized (CH) MSP142 gene fragments from 3D7 and Camp strain P. falciparum: U, cell lysates from uninduced cells; I 3hr, lysate prepared 3 hrs post induction, respectively, after induction with 0.1 mM IPTG.
Samples were separated by SDS-PAGE and stained with Coomassie Blue. Panel A; MSP142 (3D7): expression of wild type (WT) and codon harmonized MSP142 3D7.2 (CH) genes respectively. Panel B; expression of codon harmonized MSP142 Camp.2 gene. The arrows indicate the migration of MSP142.
Figure 6
Figure 6. Evaluation of expression levels for the E. coli codon optimized LSA-NRCE (3D7) and full gene codon harmonized LSA-NRCH (3D7).
Total E. coli cell lysates were separated by SDS-PAGE followed by staining with Coomassie Blue at the time of induction (U) and 3 hours post induction (I 3hr) with 0.1 mM IPTG. The arrow indicates the migration of LSA-NRC.

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