Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.     

Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain

Virtually nothing is known about the interaction of co-translationally active chaperones with nascent polypeptides and the resulting effects on peptide conformation and folding. We have explored this issue by NMR analysis of apomyoglobin N-terminal fragments of increasing length, taken as models for different stages of protein biosynthesis, in the absence and presence of the substrate binding domain of Escherichia coli Hsp70, DnaK-beta. The incomplete polypeptides misfold and self-associate under refolding conditions. In the presence of DnaK-beta, however, formation of the original self-associated species is completely or partially prevented. Chaperone interaction with incomplete protein chains promotes a globally unfolded dynamic DnaK-beta-bound state, which becomes folding-competent only upon incorporation of the residues corresponding to the C-terminal H helix. The chaperone does not bind the full-length protein at equilibrium. However, its presence strongly disfavors the kinetic accessibility of misfolding side-routes available to the full-length chain. This work supports the role of DnaK as a "holder" for incomplete N-terminal polypeptides. However, as the chain approaches its full-length status, the tendency to intramolecularly bury non-polar surface efficiently outcompetes chaperone binding. Under these conditions, DnaK serves as a "folding enhancer" by supporting folding of a population of otherwise folding-incompetent full-length protein chains.     

Protein translocation in the endoplasmic reticulum. Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins.

We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light. In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction. In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures. The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide. The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins. Both photolysis and alkaline carbonate extraction were required to produce the results. Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels. The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins. The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.     

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).     

Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro

Cotranslational protein maturation is often studied in cell-free translation mixtures, using stalled ribosome-nascent chain complexes produced by translating truncated mRNA. This approach has two limitations: (i) it can be technically challenging, and (ii) it only works in vitro, where the concentrations of cellular components differ from concentrations in vivo. We have developed a method to produce stalled ribosomes bearing nascent chains of a specified length by using a 'stall sequence', derived from the Escherichia coli SecM protein, which interacts with residues in the ribosomal exit tunnel to stall SecM translation. When the stall sequence is expressed at the end of nascent chains, stable translation-arrested ribosome complexes accumulate in intact cells or cell-free extracts. SecM-directed stalling is efficient, with negligible effects on viability. This method is straightforward and suitable for producing stalled ribosome complexes in vivo, permitting study of the length-dependent maturation of nascent chains in the cellular milieu.     

Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.     

Incomplete polypeptides are formed in vitro by premature chain termination

The mechanism of incomplete polypeptides formation during protein synthesis was studied in the wheat germ cell-free system programmed with brome mosaic virus RNA 4. The synthesis of coat protein, the complete product of RNA 4 translation, was accompanied by the appearance of polypeptides of lower molecular mass. It was shown that incomplete products are formed by translation of different lengths of RNA 4, always from the first 5' AUG codon, and were due neither to proteolysis of coat protein nor to the translation of nucleolytic fragments of mRNA. The molecular masses of incomplete products were determined and the nucleotide sequence of RNA 4 was examined in the regions where wheat germ ribosomes stop translating. It was found that they contained, on average, a slightly higher guanosine content than the total coding part of RNA 4. Translation of RNA 4 in the reticulocyte lysate resulted in a marked diminution of incomplete polypeptides. Addition of high-speed supernatant from reticulocyte lysate prevented the formation of incomplete products during translation of RNA 4 in the wheat germ system. This suggests that reticulocyte lysate contains some factor(s) which facilitate the movement of ribosomes beyond the regions where the elongation is retarded.     

Kinetics of macrolide action: the josamycin and erythromycin cases

Members of the macrolide class of antibiotics inhibit peptide elongation on the ribosome by binding close to the peptidyltransferase center and blocking the peptide exit tunnel in the large ribosomal subunit. We have studied the modes of action of the macrolides josamycin, with a 16-membered lactone ring, and erythromycin, with a 14-membered lactone ring, in a cell-free mRNA translation system with pure components from Escherichia coli. We have found that the average lifetime on the ribosome is 3 h for josamycin and less than 2 min for erythromycin and that the dissociation constants for josamycin and erythromycin binding to the ribosome are 5.5 and 11 nM, respectively. Josamycin slows down formation of the first peptide bond of a nascent peptide in an amino acid-dependent way and completely inhibits formation of the second or third peptide bond, depending on peptide sequence. Erythromycin allows formation of longer peptide chains before the onset of inhibition. Both drugs stimulate the rate constants for drop-off of peptidyl-tRNA from the ribosome. In the josamycin case, drop-off is much faster than drug dissociation, whereas these rate constants are comparable in the erythromycin case. Therefore, at a saturating drug concentration, synthesis of full-length proteins is completely shut down by josamycin but not by erythromycin. It is likely that the bacterio-toxic effects of the drugs are caused by a combination of inhibition of protein elongation, on the one hand, and depletion of the intracellular pools of aminoacyl-tRNAs available for protein synthesis by drop-off and incomplete peptidyl-tRNA hydrolase activity, on the other hand.     

Initiating translation with D-amino acids

Here we report experimental evidence that the translation initiation apparatus accepts D-amino acids ((D)aa), as opposed to only L-methionine, as initiators. Nineteen (D)aa, as the stereoisomers to their natural L-amino acids, were charged onto initiator tRNA(fMet)(CAU) using flexizyme technology and tested for initiation in a reconstituted Escherichia coli translation system lacking methionine, i.e., the initiator was reprogrammed from methionine to (D)aa. Remarkably, all (D)aa could initiate translation while the efficiency of initiation depends upon the type of side chain. The peptide product initiated with (D)aa was generally in a nonformylated form, indicating that methionyl-tRNA formyltransferase poorly formylated the corresponding (D)aa-tRNA(fMet)(CAU). Although the inefficient formylation of (D)aa-tRNA(fMet)(CAU) resulted in modest expression of the corresponding peptide, preacetylation of (D)aa-tRNA(fMet)(CAU) dramatically increased expression level, implying that the formylation efficiency is one of the critical determinants of initiation efficiency with (D)aa. Our findings provide not only the experimental evidence that translation initiation tolerates (D)aa, but also a new means for the mRNA-directed synthesis of peptides capped with (D)aa or acyl-(D)aa at the N terminus.     

Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems

Heterologous expression of a bacterial light-harvesting (LH) integral membrane protein was attempted using Escherichia coli cells and cell-free synthesis systems prepared from E. coli extracts. The alpha-apoprotein of LH1 complex from purple photosynthetic bacterium Rhodospirillum rubrum was overexpressed as a recombinant protein with a histidine (His6) tag added to the carboxyl terminus. Both of the expression systems produced alpha-apoprotein in a fully functional form as can judged by its ability to form a structural subunit with native beta-apoprotein and the pigment molecule bacteriochlorophyll a. The expression product in E. coli appears to be located in the inner cell membrane and can be almost completely extracted by 0.5% (w/v) Triton X-100. Circular dichroism measurement indicated that the expressed alpha-apoproteins from both systems had alpha-helical contents essentially identical with that of the native one. About two thirds of the alpha-apoprotein expressed in E. coli was found to have the amino terminal methionine residue modified by a formyl group. About one third of the alpha-apoprotein expressed in the cell-free system was found to be oxidized at the side chain of the amino terminal methionine residue. Functional expression of the alpha-apoprotein using the cell-free system provides an useful example for producing highly hydrophobic integral membrane proteins with relatively large quantities sufficient for biophysical and structural analysis.     

SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG1) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.Key words: cell-free protein synthesis, Fab antibody, aglycosylated antibodies, HER2, trastuzumab     

Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

We describe protein synthesis, folding and assembly of antibody fragments and full-length aglycosylated antibodies using an Escherichia coli-based open cell-free synthesis (OCFS) system. We use DNA template design and high throughput screening at microliter scale to rapidly optimize production of single-chain Fv (scFv) and Fab antibody fragments that bind to human IL-23 and IL-13α1R, respectively. In addition we demonstrate production of aglycosylated immunoglobulin G (IgG₁) trastuzumab. These antibodies are produced rapidly over several hours in batch mode in standard bioreactors with linear scalable yields of hundreds of milligrams/L over a 1 million-fold change in scales up to pilot scale production. We demonstrate protein expression optimization of translation initiation region (TIR) libraries from gene synthesized linear DNA templates, optimization of the temporal assembly of a Fab from independent heavy chain and light chain plasmids and optimized expression of fully assembled trastuzumab that is equivalent to mammalian expressed material in biophysical and affinity based assays. These results illustrate how the open nature of the cell-free system can be used as a seamless antibody engineering platform from discovery to preclinical development of aglycosylated monoclonal antibodies and antibody fragments as potential therapeutics.     

Biosynthesis of rabbit haptoglobin: chemical evidence for a single chain precursor

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the beta chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of Mr 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the alpha chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by alpha and beta chain regions, respectively.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

Partial characterizatio and proposed mode of action of inhibitory heLa cell surface polypeptides

Polypeptides removed from the HeLa cell surface by mild pronase treatment rapidly inhibit protein synthesis when added to HeLa cells or cell-free translation system derived from HeLa cells. The inhibitory activity is heat stable. Protein and carbohydrate components of these polypeptides are required for inhibition of protein synthesis in vivo and in vitro. Two peaks of activity can be recovered from polyacrylamide gels, corresponding to polypeptides with molecular weights of approximately 29 000 and 41 000. Inhibition of protein synthesis in cell-free translation systems appears to be primarily an effect on elongation of polypeptide chains, whereas in the intact cell the primary target may be polypeptide chain initiation.     

Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.