Initiation of translation at AUC, AUA and AUU codons in Escherichia coli

A truncated form of the HBL murein hydrolase, encoded by the temperate bacteriophage HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using AUC as start codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed mutagenesis, we have changed this AUC codon into AUA, AUU and AUG codons. The relative translation efficiencies for these triplets were about 5% for AUC and AUU and 7.5% for AUA compared to that of AUG codon. In the same gene arrangement E. coli beta-galactosidase was also translated at moderate efficiency using AUC as initiator.     

Initiation of translation at an upstream non-AUG codon accounting for N-terminally extended minor forms of recombinant proteins expressed in insect cells

When the 34 kDa kinase domain of human spleen tyrosine kinase (Syk-KD) was expressed as a C-terminally His-tagged protein in baculovirus-infected Sf-21 insect cells, the purified protein included two forms that migrated slightly differently in SDS-polyacrylamide gel electrophoresis. Intact mass analysis and LC-MS/MS peptide mapping showed that the major and faster-migrating product had the intended amino-acid sequence and 0-6 phosphorylations. This material accounted for about 95% of the purified protein. The minor product was Syk-KD with a 26 amino-acid N-terminal extension. The result suggested the existence of an upstream alternative site for the initiation of translation, and this proved to be an ACG codon derived from the pBacPAK9 vector used to express Syk-KD. The ACG codon was preceded and followed by Kozak-type sequence elements (a purine in the -3 position and a G in the +4 position) that would have enhanced the viability of initiation at ACG. The initiating amino-acid residue was Met for both minor and major products, and both forms of the protein were α-N-acetylated. For the minor product, protein intact mass analysis and peptide mapping both gave results in agreement with the sequence predicted from the DNA. A similar result with the same underlying cause was obtained with insect cell expression of full-length Syk. It appears that similar results are possible whenever this vector is used.     

Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

The gene coding for the key glycolytic enzyme fructose-1,6-diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.     

Effect of structure of the initiator codon on translation in E. coli

A set of plasmids carrying different initiator codons--either AUG, or GUG, or UUG, or CUG (as a control) in the hybrid gene lacIZ--was constructed by using synthetic oligonucleotides. GUG and UUG codons were demonstrated to be 2-3 times less effective than AUG in translation initiation. Furthermore, the correlation between the efficiencies of different initiator codons in translation initiation proved to vary, depending on the phase of bacterial growth. The rarely occurring usage in nature of the initiator codons GUG and UUG is supposed to be due to the particular role played by the initiator triplets in regulation of gene expression.     

Efficient suppression of the amber codon in E. coli in vitro translation system

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA(Ser(CUA)) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.     

Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein

Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.     

Initiation at AUGUG and GUGUG sequences can lead to translation of overlapping reading frames in E. coli

During initiation, the ribosome is tasked to efficiently recognize open reading frames (ORFs) for accurate and fast translation of mRNAs. A critical step is start codon recognition, which is modulated by initiation factors, mRNA structure, a Shine Dalgarno (SD) sequence and the start codon itself. Within the Escherichia coli genome, we identified more than 50 annotated initiation sites harboring AUGUG or GUGUG sequence motifs that provide two canonical start codons, AUG and GUG, in immediate proximity. As these sites may challenge start codon recognition, we studied if and how the ribosome is accurately guided to the designated ORF, with a special focus on the SD sequence as well as adenine at the fourth coding sequence position (A4). By in vitro and in vivo experiments, we characterized key requirements for unambiguous start codon recognition, but also discovered initiation sites that lead to the translation of both overlapping reading frames. Our findings corroborate the existence of an ambiguous translation initiation mechanism, implicating a multitude of so far unrecognized ORFs and translation products in bacteria.     

Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein

The pea (Pisum sativum L.) gene PSMTA has an ORF encoding a predicted protein with sequence similarity to class I metallothioneins (MTS). To examine the metal-binding properties of the PSMTA protein it has been expressed in E. coli as a carboxyterminal extension of glutathione-S-transferase (GST). Metal ions were associated with the expressed protein when purified from lysates of E. coli grown in metal supplemented media. The pH of half-dissociation of Zn, Cd and Cu ions from the recombinant fusion protein was determined to be 5.35, 3.95 and 1.45 respectively, compared with equivalent estimates of 4.50, 3.00 and 1.80 for equine renal MT.     

Optimization of an osmotic shock procedure for isolation of a protein product expressed in E. coli

Small-scale experiments were performed to evaluate the impact of several processing variables on recovery by osmotic shock of a protein product expressed in E. coli utilizing feed material manufactured at large scale. Equilibration time, type and concentration of osmotic supplements, and pH of the osmotic shock buffer were found to have significant impact on the osmotic shock recovery. Improved recovery was documented under conditions of increased equilibration time before the osmotic shock and increased Tris-HCl concentration in the osmotic shock buffer, as well as initial dispersal of cells into the osmotic shock buffer. Recovery of up to 65% was achieved under the optimized conditions.     

Efficient translation initiation dictates codon usage at gene start

The genetic code is degenerate; thus, protein evolution does not uniquely determine the coding sequence. One of the puzzles in evolutionary genetics is therefore to uncover evolutionary driving forces that result in specific codon choice. In many bacteria, the first 5–10 codons of protein‐coding genes are often codons that are less frequently used in the rest of the genome, an effect that has been argued to arise from selection for slowed early elongation to reduce ribosome traffic jams. However, genome analysis across many species has demonstrated that the region shows reduced mRNA folding consistent with pressure for efficient translation initiation. This raises the possibility that unusual codon usage is a side effect of selection for reduced mRNA structure. Here we discriminate between these two competing hypotheses, and show that in bacteria selection favours codons that reduce mRNA folding around the translation start, regardless of whether these codons are frequent or rare. Experiments confirm that primarily mRNA structure, and not codon usage, at the beginning of genes determines the translation rate.     

Coupling between codon usage, translation and protein export in Escherichia coli

Proteins destined for export via the Sec-dependent pathway are synthesized with a short N-terminal signal peptide. A requirement for export is that the proteins are in a translocationally competent state. This is a loosely folded state that allows the protein to pass through the SecYEG apparatus and pass into the periplasm. In order to maintain pre-secretory proteins in an export-competent state, there are many factors that slow the folding of the pre-secretory protein in the cytoplasm. These include cytoplasmic chaperones, such as SecB, and the signal recognition particle, which bind the pre-secretory protein and direct it to the cytoplasmic membrane for export. Recently, evidence has been published that non-optimal codons in the signal sequence are important for a time-critical early event to allow the correct folding of pre-secretory proteins. This review details the recent developments in folding of the signal peptide and the pre-secretory protein.     

Cyanobacterial metallothionein gene expressed in Escherichia coli. Metal-binding properties of the expressed protein.

The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT). To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase. The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT). E. coli expressing this gene showed enhanced (ca. 3-fold) accumulation of Zn.     

Streptococcus pneumoniae polA gene is expressed in Escherichia coli and can functionally substitute for the E. coli polA gene.

The Streptococcus pneumoniae polA+ gene was introduced into Escherichia coli on the recombinant plasmid pSM31, which is based on the pSC101 replicon. Extracts of E. coli polA5 mutants containing pSM31 showed DNA polymerase activity, indicating that the pneumococcal DNA polymerase I was expressed in the heterospecific host. Complete complementation of the E. coli polA5 mutation by the pneumococcal polA+ gene was detected in excision repair of DNA damage.     

Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene of E. coli.

We describe a method which permits the detection of exon fragments. Such DNA was cloned and expressed in the promoter proximal part of the lac Z gene of Escherichia coli. The resulting antigen-beta-galactosidase chimeras are bound to their respective antibodies fixed to polyvinyl sheets. The beta-galactosidase part of the chimera permits detection of such clones by histochemical staining. As model DNA, we used the lac I gene cleaved with HaeIII, HhaI, or HpaII. Fragments were tailed with poly(dC) and inserted into the poly(dG)-tailed promoter proximal part of the lac Z gene. Recombinant clones, isolated on lactose-agar plates, were replica-plated and lysed with chloroform. Polyvinyl sheets coated with antibody against lac repressor were placed onto the top of the lysed colonies for immunoadsorption. The immune complexes were made visible after washing by incubation with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside in buffered agar. The beta-galactosidase activity of the chimera cleaves the colourless histochemical compound to a blue dye at those positions where clones produce the antigen. In the case of the lac I gene two types of clones were isolated, carrying the NH2-terminal part of the lac repressor up to codons 27 and 75.     

Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency.

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.