Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

Abstract

Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77- to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9- to 7.1-fold or 22.5- to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3- to 8-fold or 91- to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codon-optimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

Figure 1.

Development of a codon optimization algorithm for the expression of heterologous genes in plant chloroplasts. A, Process to develop the codon optimization algorithm. Sequence data of psbA genes from 133 plant species collected from the National Center for Biotechnology Information, and their codon preferences, were analyzed. Finally, the codon optimizer was developed using Java. B, Codon preference table. Codon preference is indicated by the percentage of use for each amino acid. Black and underlined codons indicate codons that were not used when optimizing sequences due to their low usage frequency among synonymous codons (less than 5% use or, for amino acids with six synonymous codons [Leu, Ser, and Arg], the two codons used least frequently).

Figure 2.

Construction of chloroplast vectors using native or codon-optimized genes, and evaluation of homoplasmy and transgene expression. A, Lettuce or tobacco chloroplast vector maps. aadA, Aminoglycoside 3′-adenylytransferase gene; CNTB, coding sequence of cholera nontoxic B subunit; FVIII HC, factor 8 heavy chain native (N) or codon optimized (C^N) using the new algorithm; PpsbA, promoter and 5′ UTR of the psbA gene; Prrn, rRNA operon promoter; SB-P, BamHI fragment; TpsbA, 3′ UTR of the psbA gene; trnA, alanyl-tRNA; trnI, isoleucyl-tRNA. B and C, Southern-blot analysis of homoplasmic lines. Total genomic DNA (3 µg) from untransformed (UT), native (N), or codon-optimized CNTB-FVIII HC (new algorithm; C^N) was digested with HindIII and separated on a 0.8% agarose gel, blotted onto a Nytran membrane, and probed with a BamHI fragment. Lanes 1 to 4 show four independent transplastomic lines. L.s., Lactuca sativa. D, Comparison of the expression level of CNTB-VP1 between transplastomic lines expressing the native (N) or codon-optimized genes using the old (C^O) or new (C^N) algorithm. Total extracted proteins were loaded as indicated protein concentrations and were probed with anti-CNTB antibody. CNTB, Standard protein of cholera nontoxic B subunit; IDV, integrated density values; N.t., Nicotiana tabacum.

Figure 3.

Quantitation of native or codon-optimized CNTB-FVIII HC or CNTB-VP1 gene expression using western blots. Extracted leaf proteins were resolved on gradient (4%–20%) SDS-PAGE and probed with anti-CNTB antibody (1:10,000). For a loading control, the same membranes were stripped and reprobed with anti-RbcL antibody (1:5,000). A, Lettuce leaf protein extracts (5 or 10 μg) expressing CNTB-FVIII HC or untransformed. For loading controls, Ponceau S staining of membrane prior to western blot or reprobed blot with the large subunit of Rubisco (RbcL) is provided. B, Serial dilution of the native (5–20 μg) or codon-optimized (1–4 μg) CNTB-FVIII HC lettuce leaf extracts. C, Serial dilution of the native (2–8 μg) or codon-optimized (0.1–0.4 μg) CNTB-VP1 tobacco leaf extracts. C^O or C^N, Codon optimized with old algorithm (C^O) or new algorithm (C^N); L.s., Lactuca sativa; N, native sequence; N.t., Nicotiana tabacum; UT, untransformed wild type.

Figure 4.

Northern analysis of transplastomic lines. Transgene transcripts of CNTB-FVIII HC (A) or CNTB-VP1 (B) were probed with 200 bp of lettuce psbA 5ʹ UTR (for FVIII HC) or tobacco psbA 5ʹ UTR (for VP1) regulatory sequences. Bottom and top arrowheads represent the endogenous psbA gene and CNTB-FVIII or CNTB-VP1 transgene, respectively. Ethidium bromide (EtBr)-stained gels are included for the evaluation of equal loading. C^N, Codon-optimized sequence using the new algorithm; N, native sequence; UT, untransformed wild type.

Figure 5.

PRM mass spectrometry analysis of CNTB-FVIII and CNTB-VP1 proteins at N- to C-terminal protein sequences. The y axis shows molarity (fmol on column) of peptides from CNTB-FVIII HC or CNTB-VP1 in codon-optimized or native genes. CNTB: peptide 1, IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII: peptide 4, FDDDNSPSFIQIR; peptide 5, WTVTVEDGPTK; peptide 6, YYSSFVNMER. The median of four technical replicates is presented for each sample. Circles represent native sequences, and squares represent codon-optimized (c.o.) sequences using the new algorithm. CV, Coefficient of variation.

Figure 6.

Fold change (increase) of CNTB-FVIII HC or CNTB-VP1 proteins based on targeted mass spectrometry analysis of CNTB and HC peptides. The reported data represent medians of the results from six and three peptides from CNTB-FIII HC (A) and CNTB-VP1 (B), respectively. The y axis represents the fold change increase (based on measured fmol on column) of peptides from plant materials expressing genes codon optimized using the new algorithm (C^O) with respect to plant materials expressing native sequence (N). CNTB: peptide 1, IFSYTESLAGK; peptide 2, IAYLTEAK; peptide 3, LCVWNNK. FVIII HC: peptide 4, FDDDNSPSFIQIR; peptide 5, WTVTVEDGPTK; peptide 6, YYSSFVNMER. SIS-normalized values represent fold change as a ratio to each spiked SIS peptide. Housekeeping (HK) protein normalization values represent fold change as a normalized ratio to Rubisco large or small subunit and ATP synthase CF1 β-subunit protein peptides. For peptide ratio results for CNTB-FVIII and CNTB-VP1, see Supplemental Data Set S1.

Figure 7.

Ribosome profiling data from transplastomic plants expressing native and codon-optimized VP1 or FVIII HC. Read coverage for native transgenes (N), codon-optimized transgenes with new algorithm (C^N), and the endogenous psbA and rbcL genes are displayed with the Integrated Genome Viewer. A, Data from tobacco leaves expressing native and codon-optimized VP1 transgenes. Asterisks mark each pair of consecutive Ala codons in the data from the native line. The + symbol marks three consecutive Ala codons. Many strong ribosome pause sites in the plants expressing native VP1 map to paired Ala codons, whereas this is not observed in the codon-optimized line. Triangles mark each pair of consecutive Ser codons in the codon-optimized line. A major ribosome stall maps to a region harboring five closely spaced Ser codons in the codon-optimized VP1 gene. nt, Nucleotides. B, Data from lettuce plants expressing the native and codon-optimized FVIII HC transgenes. A major ribosome stall in the native FVIII HC gene maps to a pair of adjacent CTC Leu codons, a codon that is not used in the native psbA gene. Ribosome footprint coverage is much more uniform on the codon-optimized transgene. C, Absolute and relative ribosome footprints counts.