Preferential codons enhancing the expression level of human beta-defensin-2 in recombinant Escherichia coli

Human beta-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli.     

Termination of translation in bacteria may be modulated via specific interaction between peptide chain release factor 2 and the last peptidyl-tRNA(Ser/Phe).

The 5' context of 671 Escherichia coli stop codons UGA and UAA has been compared with the context of stop-like codons (UAC, UAU and CAA for UAA; UGG, UGC, UGU and CGA for UGA). We have observed highly significant deviations from the expected nucleotide distribution: adenine is over-represented whereas pyrimidines are under-represented in position -2 upstream from UAA. Uridine is over-represented in position -3 upstream from UGA. Lysine codons are preferable immediately prior to UAA. A complete set of codons for serine and the phenylalanine UUC codon are preferable immediately 5' to UGA. This non-random codon distribution before stop codons could be considered as a molecular device for modulation of translation termination. We have found that certain fragment of E. coli release factor 2 (RF2) (amino acids 93-114) is similar to the amino acid sequences of seryl-tRNA synthetase (positions 10-19 and 80-93) and of beta (small) subunit (positions 72-94) of phenylalanyl-tRNA synthetase from E. coli. Three-dimensional structure of E. coli seryl-tRNA synthetase is known [1]: Its N-terminus represents an antiparallel alpha-helical coiled-coil domain and contains a region homologous to RF2. On the basis of the above-mentioned results we assume that a specific interaction between RF2 and the last peptidyl-tRNA(Ser/Phe) occurs during polypeptide chain termination in prokaryotic ribosomes.     

Expression of mature pulmonary surfactant-associated protein B (SP-B) in Escherichia coli using truncated human SP-B cDNAs

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)     

5' contexts of Escherichia coli and human termination codons are similar.

The nearest 5' context of 2559 human stop codons was analysed in comparison with the same context of stop-like codons (UGG, UGC, UGU, CGA for UGA; CAA, UAU, UAC for UAA; and UGG, UAU, UAC, CAG for UAG). The non-random distribution of some nucleotides upstream of the stop codons was observed. For instance, uridine is over-represented in position -3 upstream of UAG. Several codons were shown to be over-represented immediately upstream of the stop codons: UUU(Phe), AGC(Ser), and the Lys and Ala codon families before UGA; AAG(Lys), GCG(Ala), and the Ser and Leu codon families before UAA; and UCA(Ser), AUG(Met), and the Phe codon family before UAG. In contrast, the Thr and Gly codon families were under-represented before UGA, while ACC(Thr) and the Gly codon family were under-represented before UAG and UAA respectively. In an earlier study, uridine was shown to be over-represented in position -3 before UGA in Escherichia coli [Arkov,A.L., Korolev,S.V. and Kisselev,L.L. (1993) Nucleic Acids Res., 21,2891-2897]. In that study, the codons for Lys, Phe and Ser were shown to be over-represented immediately upstream of E. coli stop codons. Consequently, E. coli and human termination codons have similar 5' contexts. The present study suggests that the 5' context of stop codons may modulate the efficiency of peptide chain termination and (or) stop codon readthrough in higher eukaryotes, and that the mechanisms of such a modulation in prokaryotes and higher eukaryotes may be very similar.     

Mutation of the Secys residue 266 in human type 2 selenodeiodinase alters 75Se incorporation without affecting its biochemical properties.

Type 2 deiodinase (D2) is a low Km iodothyronine deiodinase that metabolizes thyroxine (T4) to the active metabolite T3. We have recently shown that the cDNA for the human D2 coding region contains two in-frame selenocysteine (TGA) codons. The 3' TGA is seven codons 5' to a universal stop codon, TAA. The human D2 enzyme, transiently expressed in HEK-293 cells, can be in vivo labeled with 75Se as a doublet of approximately 31 kDa. This doublet is consistent with the possibility that the carboxy-terminal TGA codon can either encode selenocysteine or function as a stop codon. To test this hypothesis we mutagenized the second selenocysteine codon to a cysteine (TGC) or to an unambiguous stop codon (TAA). While the selenium incorporation pattern is different between the wild-type and mutant proteins, the deiodination properties of the enzyme are not affected by mutating the 3'TGA codon. Thus, we conclude that neither this residue nor the remaining seven carboxy-terminal amino acids are critical for the deiodination process.     

Identification of a heat-shock pseudogene from Caenorhabditis elegans

While characterizing the hsp70 gene family from Caenorhabditis elegans we encountered an unusual member of this family. Sequence data reveal that the hsp-2ps gene is a pseudogene of the constitutively expressed, heat-inducible hsp-1 gene. Two stop codons generated near the 5' end of the sequence as well as several frameshift mutations and a large internal deletion confirm the identification of hsp-2ps as a pseudogene. The nucleotide substitution rate of the third codon position was twice that of the first and second codon positions, suggesting that the hsp-2ps gene was nonfunctional since the time of the duplication event. The hsp-2ps gene duplicates a region of the hsp-1 gene that lies exclusively within the transcribed region and retains the introns. We feel that the hsp-2ps gene was produced by a transpositional duplication event, which occurred approximately 8.5 million years ago.     

Regularities of the nucleotide sequence at the 5'-end of the codon in Escherichia coli genes

The frequencies of occurrence of nucleotides at the 5' side of codons have been determined in highly and weakly expressed genes from E. coli. Significant constraints on the nucleotide 5' to some codons were found in highly expressed genes. Certain rules of synonymous codon usage depending on the amino acid 3' of the codon were established. E. g., codon possessing quanosine in the third position (NNG) are preferred over NNA if the next amino acid is lysine (P less than 10(-5)). On the other hand, rules of synonymous codon usage in relation to 5' flanking nucleotide were found. For example, when coding for aspartic acid, GAC codon is preferred over GAU (P less than 0.001) if uridine is 5' to codon and on the contrary GAU is favoured (P less than 0.0001) if quanosine is at the 5' side of aspartic acid codon. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Cost minimization of ribosomal frameshifts

Properties of mRNA leading regions that modulate protein synthesis are little known (besides effects of their secondary structure). Here I explore how coding properties of leading regions may account for their disparate efficiencies. Trinucleotides that form off frame stop codons decrease costs of ribosomal slippages during protein synthesis: protein activity (as a proxy of gene expression, and as measured in experiments using artificial variants of 5' leading sequences of beta galactosidase in Escherichia coli) increases proportionally to the number of stop motifs in any frame in the 5' leading region. This suggests that stop codons in the 5' leading region, upstream of the recognized coding sequence, terminate eventual translations that sometimes start before ribosomes reach the mRNA's recognized start codon, increasing efficiency. This hypothesis is confirmed by further analyses: mRNAs with 5' leading regions containing in the same frame a start preceding a stop codon (in any frame) produce less enzymatic activity than those with the stop preceding the start. Hence coding properties, in addition to other properties, such as the secondary structure of the 5' leading region, regulate translation. This experimentally (a) confirms that within coding regions, off frame stops increase protein synthesis efficiency by early stopping frameshifted translation; (b) suggests that this occurs for all frames also in 5' leading regions and that (c) several alternative start codons that function at different probabilities should routinely be considered for all genes in the region of the recognized initiation codon. An unknown number of short peptides might be translated from coding and non-coding regions of RNAs.     

同义密码子用语的位置依赖

研究了在大肠杆菌编码区不同位置上的同底密码子用语,发现许多氨基酸的密码子用语在转译起始区有显著的变化,仅有少数氨基酸在转译区有较弱的变化,由于密码子用语与基因表达关系密切。这些结果与实验发现的编码区5‘端密码子用对表达的重要性是一致的。更进一步的结果还暗示了哪些密码子在特定位置的使用可能会影响基因表达。     

Translational features of human alpha 2b interferon production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

Characterization and heterospecific expression of cDNA clones of genes in the maize GSH S-transferase multigene family

We have isolated from a constructed lambda gt11 expression library two classes of cDNA clones encoding the entire sequence of the maize GSH S-transferases GST I and GST III. Expression of a full-length GST I cDNA in E. coli resulted in the synthesis of enzymatically active maize GST I that is immunologically indistinguishable from the native GST I. Another GST I cDNA with a truncated N-terminal sequence is also active in heterospecific expression. Our GST III cDNA sequence differs from the version reported by Moore et al. [Moore, R. E., Davies, M. S., O'Connell, K. M., Harding, E. I., Wiegand, R. C., and Tiemeier, D. C. (1986) Nucleic Acids Res. 14:7227-7235] in eight reading frame shifts which result in partial amino acid sequence conservation with the rat GSH S-transferase sequences. The GST I and GST III sequences share approximately 45% amino acid sequence homology. Both the GST I and the GST III mRNAs contain different repeating motifs in front of the initiation codon ATG. Multiple poly(A) addition sites have been identified for these two classes of maize GSH S-transferase messages. Genomic Southern blotting results suggest that both GST I and GST III are present in single or low copies in the maize (GT112 RfRf) genome.     

Expression of the transglutaminase gene in Escherichia coli.

1. The cDNA gene coding for the enzyme transglutiminase (EC 2.3.2.13) was cloned into the pUC18 oriented for expression from the lac promoter. 2. DNA sequencing of the 5' end showed that the cDNA was missing the sequence coding of the N-terminal 30 amino acids. 3. The truncated gene was then cloned into pKK233-2, and the recombinant product was produced in Escherichia coli. 4. A gene construct coding for the complete protein was generated by inserting an oligonucleotide for the missing 30 amino acids into the Eco RI site of the pUC18 clone. 5. A consensus Shine-Dalgarno sequence and translational start codon were positioned at the 5' end of the linker. 6. Immunoblotting experiments of E. coli JM105(pUC18-TGase) indicated the expression of the transglutaminase gene. 7. The cell lysate as well as the partially purified transglutaminase showed no detectable enzyme activity.     

Constraints on codon context in Escherichia coli genes. Their possible role in modulating the efficiency of translation

The constraints on nucleotide sequences of highly and weakly expressed genes from Escherichia coli have been analysed and compared. Differences in synonymous codon spectra in highly and weakly expressed genes lead to different frequencies of nucleotides (in the first and third codon positions) and dinucleotides in the two groups of genes. It has been found that the choice of synonymous codons in highly expressed genes depends on the nucleotides adjacent to the codon. For example, lysine is preferably encoded by the AAA codon if guanosine is 3' to the lysine codon (AAA-G, P less than 10(-9)). And, on the contrary, AAG is used more often than AAA (P less than 0.001) if cytidine is 3' adjacent to lysine. Guanosine occurs more frequently than adenosine 5' to all the lysine codons (AAR, P less than 10(-5), i.e. NNG codons are preferred over the synonymous NNA codons 5' to the positions of lysine in the genes. The context effect was observed in nonsense and missense suppression experiments. Therefore, a hypothesis has been suggested that the efficiency of translation of some codons (for which the constraints on the adjacent nucleotides were found) can be modulated by the codon context. The rules for preferable synonymous codon choice in highly expressed genes depending on the nucleotides surrounding the codon are presented. These rules can be used in the chemical synthesis of genes designed for expression in E. coli.     

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

Expression of enterovirus 70 capsid protein VP1 in Escherichia coli

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.     

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.