Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.     

Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

Na⁺-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium.     

Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase

We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.     

A conformational switch from a closed apo- to an open holo-form equips the acyl carrier protein for acyl chain accommodation

Acyl carrier proteins (ACPs) play crucial roles in the biosynthesis of fatty acids, non-ribosomal polypeptides and polyketides. The three-dimensional NMR structure of Leishmania major holo-LmACP, belonging to the type II pathway, has been reported previously, but the structure of its apo-form and its conformational differences with the holo-form remain to be explored. Here we report the crystal structures of apo-LmACP (wild-type and S37A mutant) at 2.0 Å resolution and compare their key features with the structures of holo-LmACP (wild-type) and other type II ACPs from Escherichia coli and Plasmodium falciparum. The crystal structure of apo-LmACP, which is homologous to other type II ACPs, displays some key structural rearrangements as compared to its holo-structure. Contrary to holo-form, which exists predominantly as a monomer, the apo-form exists as a mixture of monomeric and dimeric population in solution. In contrast to the closed structure of apo-LmACP, holo-LmACP structure was observed in an open conformation as a result of reorganization of specific helices and loops. We propose that the structural changes exhibited by LmACP occur due to the attachment of the phosphopantetheine arm and may be a prerequisite for the initiation of fatty acid synthesis. The movement of helix 3 may also play a role in the dissociation of holo-LmACP from its cognate enzymes of the FAS II pathway.     

Enhanced protein stability through minimally invasive,direct, covalent,and site‐specific immobilization

Bioconjugating protein to nonbiological surfaces is an essential component of many promising biotechnologies impacting diverse applications such as medical diagnostics, biocatalysis, biohazard detection, and proteomics. However, to enable the widespread economical use of immobilized‐protein technologies, long‐term stability, and reusability is essential. To enhance protein stability in harsh conditions, herein we report a minimally invasive and covalent bioconjugation that enables precise control of the immobilization location at potentially any surface‐accessible location where the incorporated unnatural amino acid does not impact protein structure and function. Specifically, the PRECISE system is introduced where a uniquely reactive unnatural amino acid was incorporated site‐specifically at a prespecified location in GFP using cell‐free protein synthesis. The GFP was then directly and covalently attached to superparamagnetic beads by the unnatural amino acid in a single click reaction. The immobilized GFP was probed for retained activity and stability under harsh conditions including freeze‐thaw cycling and incubation in urea at elevated temperatures. The immobilized GFP was more stable compared to unattached protein in all cases and for all durations observed. The enhanced stability of the immobilized protein is a promising step towards long‐term protein stability for biocatalysis and other immobilized‐protein applications. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

Bioconjugation of L-3,4-dihydroxyphenylalanine containing protein with a polysaccharide

We describe the simple bioconjugation strategy in combination of periodate chemistry and unnatural amino acid incorporation. The residue specific incorporation of 3,4-dihydroxy-l-phenylalanine can alter the properties of protein to conjugate into the polymers. The homogeneously modified protein will yield quinone residues that are covalently conjugated to nucleophilic groups of the amino polysaccharide. This novel approach holds great promise for widespread use to prepare protein conjugates and synthetic biology applications.     

Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.     

The primary structure of chicken liver cytochrome b5 deduced from the DNA sequence of a cDNA clone

A cDNA clone encoding the chicken liver cytochrome b5 was isolated by probing a library with synthetic oligonucleotides based on a partial amino acid sequence of the protein. Determination of the DNA sequence indicated a 414-nucleotide open reading frame which encodes a 138-amino acid residue polypeptide. The open reading frame contains 6 amino acids at the amino terminus which were not present on any of the cytochrome b5 polypeptides for which the amino acid sequence has been determined directly, suggesting that the protein is proteolytically processed to the mature form. The results of genomic Southern analysis were consistent with the presence of two structurally different genes in the chicken genome, raising the possibility that the soluble and membrane-bound forms of the protein are the products of separate genes.     

Cleavage of the N-terminal formylmethionine residue from a bacteriophage coat protein in vitro

Studies were made of the N-terminal formylmethionine content of nascent and complete coat protein of bacteriophage Qβ synthesized in an Escherichia coli cell-free system. Under normal conditions of cell-free protein synthesis the formylmethionine residue was retained by all the nascent chains but by only about 50% of the completed coat protein molecules. If 2-mercaptoethanol was omitted from the cell-free system, the formylmethionine residue was cleaved during the course of peptide chain elongation. All nascent peptides which contained fewer than 40±5 amino acids retained the formylmethionine residue. Thereafter, the proportion of nascent peptides lacking the residue increased with peptide length to about 70% for nearly full length nascent peptides and complete released coat protein molecules.     

High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA(CUA)(opt) and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyano-phenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.     

Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid

The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.     

Sequence specificity and efficiency of protein N-terminal methionine elimination in wheat-embryo cell-free system

Recent improvements in wheat-embryo cell-free translation resulted in a highly productive system for protein preparation. To clarify N-terminal processing of the cell-free system in a preparative-scale (> mg protein product per ml), 20 mutant variants of maltose-binding protein (MalE), each having a different penultimate residue in the sequence Met-Xaa-Ile-Glu-, and 20 glutathione S-transferase (GST) variants, having Met-Xaa-Pro-Ile-sequence, were designed and synthesized. The MalE and GST proteins were purified by amylose-resin and glutathione columns, respectively, followed by analysis of their N-terminal sequences. These investigations revealed that sequence specificity and efficiency of the N-terminal Met (N-Met) elimination in the cell-free system are similar to those reported from investigations in cellular systems or in the wheat-embryo cell-free protein expression system in analytical scale (approximately 10 microg protein product per ml). Cleavage of the N-Met is basically determined by the penultimate amino acid in the polypeptide sequence. In the case of MalE, the cleavage was efficient when the penultimate residue was Ala, Cys, Gly, Pro, Ser or Thr. But, in the case of GST with Pro as the antepenultimate residue, the efficiency was significantly reduced when the penultimate residue was Gly or Thr. We also confirmed that substitution of the antepenultimate residue in MalE to Pro drastically reduced the efficiency of N-Met cleavage when the penultimate residue was Ala, Gly, Pro, Ser or Thr, indicating inhibitory effects of antepenultimate residue Pro on N-Met elimination. These results clarified sequence-specific functions of the endogenous N-terminal processing machinery in the scaled-up wheat-embryo cell-free translation system.     

Site-selective post-translational modification of proteins using an unnatural amino acid, 3-azidotyrosine

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNA(Tyr)/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins.     

Detection of co- and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system

To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [(3)H]myristic acid followed by SDS-PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [(3)H]leucine, [(3)H]myristic acid, and [(35)S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein.     

Development of a rapid and productive cell-free protein synthesis system

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.     

Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Cell-free complements in vivo expression of the E. coli membrane proteome

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.