Optimizing the Semisynthesis towards glycosylated interferon-ß-polypeptide by utilizing bacterial protein expression and chemical modification.

The synthesis of a sufficient amount of homogeneous glycoprotein is of great interest because natural glycoproteins show considerable heterogeneity in oligosaccharide structures, making the studies on glycan structure-function relationship difficult. Herein, we report optimized methods that can accelerate the semisynthesis of homogeneous glycoproteins based on recombinant expression and chemical conversion. Peptide thioesters and peptides with Cys residues at their N-terminals are necessary intermediates to perform native chemical ligation. We successfully performed thioesterification for a peptide prepared in E. coli via Cys-cyanylation at its C-terminal followed by hydrazinolysis and acidic thiolysis. These optimized conditions could tolerate an acid labile Thz protected Cys at the N-terminal of a peptide-hydrazide and specific cyanylation of the C-terminal Cys to yield a peptide thioester. To reduce the amount of precious oligosaccharide that is required in the conventional SPPS method, an improved liquid phase glycopeptide coupling was also optimized in a good yield (46% over four steps). Lastly, chemoselective protection of the internal cysteines and activation of the N-terminal cysteine were optimized toward a long peptide prepared in E. coli. By using these strategies, a full-length interferon-ß glycosyl polypeptide as a model was successfully obtained.

Plasmid-mediated colistin resistance and ESBL production in Escherichia coli from clinically healthy and sick pigs.

This study aimed to determine the percentage of colistin resistant and ESBL-producing Escherichia coli from clinically sick and healthy pigs and understand the molecular mechanisms underlying colistin resistance and ESBL production. A total of 454 E. coli isolates from healthy pigs (n = 354; piglets, n = 83; fattening pigs, n = 142 and sows, n = 100) and sick pigs (n = 100) were examined for antimicrobial susceptibility, chromosomal and plasmid-mediated colistin resistance mechanisms and ESBL genes. The healthy (41%) and sick pig (73%) isolates were commonly resistant to colistin. Three mcr genes including mcr-1 (10.4%), mcr-2 (1.1%) and mcr-3 (45%) were detected, of which mcr-3 was most frequently detected in the healthy (33%) and sick pig (57%) isolates. Coexistence of mcr-1/mcr-3 and mcr-2/mcr-3 was observed in piglets (23%), fattening pig (3.5%) and sick pig (13%) isolates. Three amino acid substitutions including E106A and G144S in PmrA and V161G in PmrB were observed only in colistin-resistant isolates carrying mcr-3. The percentage of ESBL-producing E. coli was significantly higher in the sick pigs (44%) than the healthy pigs (19.2%) (P = 0.00). The blaCTX-M group was most prevalent (98.5%), of which blaCTX-M-14 (54.5%) and blaCTX-M-55 (42.9%) were predominant. The blaTEM-1 (68.8%) and blaCMY-2 (6.3%) genes were identified in ESBL-producers. All ESBL producers were multidrug resistant and the majority from piglets (97%), fattening pigs (77.3%) and sick pigs (82%) carried mcr gene (s). ESBL producers from piglets (n = 5) and sick pig (n = 1) simultaneously transferred blaTEM-1 (or blaCTX-M-55) and mcr-3 to Salmonella. In conclusion, pigs are important reservoirs of colistin-resistant E. coli that also produced ESBLs, highlighting the need for prudent and effective use of antimicrobials in pigs and other food-producing animals.

Co-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor.

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Sense codon reassignment enables viral resistance and encoded polymer synthesis.

It is widely hypothesized that removing cellular transfer RNAs (tRNAs)-making their cognate codons unreadable-might create a genetic firewall to viral infection and enable sense codon reassignment. However, it has been impossible to test these hypotheses. In this work, following synonymous codon compression and laboratory evolution in Escherichia coli, we deleted the tRNAs and release factor 1, which normally decode two sense codons and a stop codon; the resulting cells could not read the canonical genetic code and were completely resistant to a cocktail of viruses. We reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids. Notably, we demonstrate the facile reprogramming of our cells for the encoded translation of diverse noncanonical heteropolymers and macrocycles.

Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli.

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

In Vitro Protein Synthesis in Semipermeable Artificial Cells.

A novel cell free protein synthesis (CFPS) system utilizing layer-by-layer (LbL) polymer assembly was developed to reduce the operational cost of conventional CFPS. This yielded an encapsulated cell system, dubbed "eCells", that successfully performs in vitro CFPS and allows cost-effective incorporation of noncanonical amino acids into proteins. The use of eCells in CFPS circumvents the need for traditional cell lysate preparation and purification of amino acyl-tRNA synthetases (aaRS) while still retaining the small scale of an in vitro reaction. eCells were found to be 55% as productive as standard dialysis CFPS at 13% of the cost. The reaction was shown to be scalable over a large range of reaction volumes, and the crowding environment in eCells confers a stabilizing effect on marginally stable proteins, such as the pyrrolysl tRNA synthetase (PylRS), providing a means for their application in in vitro protein expression. Photocaged-cysteine (PCC) and Nε-(tert-butoxycarbonyl)-l-lysine (Boc-lysine) were incorporated into Peptidyl-prolyl cis-trans isomerase B (PpiB) using small amounts of ncAA with an adequate yield of protein. Fluorescent activated cell sorting (FACS) was used to demonstrate the partition of the lysate within the eCells in contrast to standard one pot cell lysate-based methods.

Consideration on Efficient Recombinant Protein Production: Focus on Substrate Protein-Specific Compatibility Patterns of Molecular Chaperones.

Expression of recombinant proteins requires at times the aid of molecular chaperones for efficient post-translational folding into functional structure. However, predicting the compatibility of a protein substrate with the right type of chaperone to produce functional proteins is a daunting issue. To study the difference in effects of chaperones on His-tagged recombinant proteins with different characteristics, we performed in vitro proteins expression using Escherichia coli overexpressed with several chaperone 'teams': Trigger Factor (TF), GroEL/GroES and DnaK/DnaJ/GrpE, alone or in combinations, with the aim to determine whether protein secondary structure can serve as predictor for chaperone success. Protein A, which has a helix dominant structure, showed the most efficient folding with GroES/EL or TF chaperones alone, whereas Protein B, which has less helix in the structure, showed a remarkable effect on the DnaK/J/GrpE system alone. This tendency was also seen with other recombinant proteins with particular properties. With the chaperons' assistance, both proteins were synthesized more efficiently in the culture at 22.5 °C for 20 h than at 37 °C for 3 h. These findings suggest a novel avenue to study compatibility of chaperones with substrate proteins and optimal culture conditions for producing functional proteins with a potential for predictive analysis of the success of chaperones based on the properties of the substrate protein.

Rapid detection of extended spectrum ß-lactamase producing Escherichia coli isolated from fresh pork meat and pig cecum samples using multiplex recombinase polymerase amplification and lateral flow strip analysis.

The emergence and dissemination of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli is a global health issue. Food-producing animals, including pigs, are significant reservoirs of antimicrobial resistance (AMR), which can be transmitted to humans. Thus, the rapid detection of ESBLs is required for efficient epidemiological control and treatment. In this study, multiplex recombinase polymerase amplification (RPA) combined with a single-stranded tag hybridization chromatographic printed-array strip (STH-PAS), as a lateral flow strip assay (LFA), was established for the rapid and simultaneous detection of multiple bla genes in a single reaction. Visible blue lines, indicating the presence of the blaCTX-M, blaSHV, and blaOXA genes, were observed within 10 min by the naked eye. The limit of detection of all three genes was 2.5 ng/25 µL, and no cross-reactivity with seven commensal aerobic bacteria was observed. A total of 93.9% (92/98) and 96% (48/50) of the E. coli isolates from pork meat and fecal samples, respectively, expressed an ESBL-producing phenotype. Nucleotide sequencing of the PCR amplicons showed that blaCTX-M was the most prevalent type (91.3-95.83%), of which the main form was blaCTX-M-55. The sensitivity and specificity of the RPA-LFA were 99.2% and 100%, respectively, and were in almost perfect agreement (κ = 0.949-1.000) with the results from PCR sequencing. Thus, the RPA-LFA is a promising tool for rapid and equipment-free ESBL detection and may facilitate clinical diagnosis in human and veterinary medicine, as well as AMR monitoring and surveillance.

Application of Dual Promoter Expression System for the Enhanced Heparosan Production in Bacillus megaterium.

Heparosan, a capsular polysaccharide synthesized by certain pathogenic bacteria, is a promising precursor for heparin production. Heparosan production is catalyzed by the formation of KfiC-KfiA complex and the subsequent action of KfiC and KfiA proteins. Polycistronic expression of kfiC and kfiA in Bacillus megaterium yielded an unbalanced expression of KfiC and KfiA proteins resulted in decreased heparosan production. In this study, dual promoter plasmid system was constructed to increase the expression levels of KfiC and KfiA proteins. Dual promoter plasmid system along with UDP-glucuronic acid pathway overexpression (CADuet-DB) increased the heparosan production to 203 mg/L in shake flask experiments. Batch fermentation of strain CADuet-DB under controlled conditions yielded a maximum heparosan concentration of 627 mg/L, which is 59% higher than strain CA-DB. A modified logistic model is applied to describe the kinetics of heparosan production and biomass growth. Fed batch fermentation resulted in 3-fold enhancement in heparosan concentration (1.96 g/L), compared to batch fermentation. Nuclear magnetic resonance analysis revealed that heparosan from strain CADuet-DB was similar to Escherichia coli K5 heparosan. These results suggested that dual promoter expression system is a promising alternative to polycistronic expression system to produce heparosan in B. megaterium.

OmpR coordinates the expression of virulence factors of Enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans.

Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.

Efficient production of a functional G protein-coupled receptor in E. coli for structural studies.

G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey ß1-adrenergic receptor (ß1AR) uniformly or selectively labeled in 15N or 2H,15N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2-0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-ß1AR mutant also comprises the two native tyrosines Y5.58 and Y7.53, which enable G protein binding. High-quality 1H-15N TROSY spectra were obtained for E. coli-expressed YY-ß1AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. RESULTS: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. DISCUSSION: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Optimization of Cephalosporin C Acylase Expression in Escherichia coli by High-Throughput Screening a Constitutive Promoter Mutant library.

Cephalosporin C acylase (CCA) is capable of catalyzing cephalosporin C (CPC) to produce 7-aminocephalosporanic acid (7-ACA), an intermediate of semi-synthetic cephalosporins. Inducible expression is usually used for CCA. To improve the efficiency of CCA expression without gene induction, three recombinant strains regulated by constitutive promoters BBa_J23105, PLtetO1, and tac were constructed, respectively. Among them, BBa_J23105 was the best promoter and its mutant libraries were established using saturation mutagenesis. In order to obtain the mutants with enhanced activity, a high-throughput screening method based on flow cytometric sorting techniques was developed by using green fluorescent protein (GFP) as the reporter gene. A series of mutants were screened at 28 °C, 200 rpm, and 24-h culture condition. The study of mutants showed that the enzyme activity, fluorescence intensity, and promoter transcriptional strength were positively correlated. The enzyme activity of the optimal mutant obtained by screening reached 12772 U/L, 3.47 times that of the original strain.

Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

Expression and Purification of the VpDef Defensin in Escherichia coli using the Small Metal-Binding Proteins CusF3H+ and SmbP.

BACKGROUND: The heterologous production of antimicrobial peptides in bacterial models can produce insoluble proteins due to the lack of proper folding. Fusion proteins have been used to increase the expression and solubility of these types of proteins with varying degrees of success. OBJECTIVES: Here, we demonstrate the use of the small metal-binding proteins CusF3H+ (9.9kDa) and SmbP (9.9kDa) as fusion partners for the soluble expression of the bioactive antimicrobial peptide VpDef(6.9 kDa) in Escherichia coli. METHODS: The recombinant VpDef (rVpDef) peptide was expressed as a translational fusion with CusF3H+ and SmbP in Escherichia coli SHuffle under different small-scale culture conditions. The best conditions were applied to 1-liter cultures, with subsequent purification of the recombinant protein through IMAC chromatography. The recombinant protein was digested using enterokinase to liberate the peptide from the fusion protein, and a second IMAC chromatography step removed the fusion protein. The purified peptide was tested against two Gram-positive and two Gram-negative bacteria. RESULTS: The use either of CusF3H+ or of SmbP results in recombinant proteins that are found in the soluble fraction of the bacterial lysate; these recombinant proteins are easily purified through IMAC chromatography, and rVpDef is readily separated following enterokinase treatment. The purified rVpDef peptide exhibits antimicrobial properties against both Gram-positive and Gram-negative. CONCLUSION: Use of the fusion proteins CusF3H+ and SmbP results in production of a soluble recombinant protein containing the antimicrobial peptide rVpDef that is correctly folded and that retains its antimicrobial properties once purified.

Enhancing n-Butanol Tolerance of Escherichia coli by Overexpressing of Stress-Responsive Molecular Chaperones.

Microbial tolerance to organic solvents is critical for efficient production of biofuels. In this study, n-butanol tolerance of Escherichia coli JM109 was improved by overexpressing of genes encoding stress-responsive small RNA-regulator, RNA chaperone, and molecular chaperone. Gene rpoS, coding for sigma S subunit of RNA polymerase, was the most efficient in improving n-butanol tolerance of E. coli. The highest OD600 and the specific growth rate of JM109/pQE80L-rpoS reached 1.692 and 0.144 h-1 respectively at 1.0% (v/v) n-butanol. Double and triple expression of molecular chaperones rpoS, secB, and groS were conducted and optimized. Recombinant strains JM109/pQE80L-secB-rpoS and JM109/pQE80L-groS-secB-rpoS exhibited the highest n-butanol tolerance, with specific growth rates of 0.164 and 0.165 h-1, respectively. Membrane integrity, potentials, and cell morphology analyses demonstrated the high viability of JM109/pQE80L-groS-secB-rpoS. This study provides guidance on employing various molecular chaperones for enhancing the tolerance of E. coli against n-butanol.

Soluble Expression, One-Step Purification and Characterization of Recombinant Human Growth Hormone Fused with ompA3 in Escherichia coli.

BACKGROUND: Human growth hormone (hGH) is the first recombinant protein approved for the treatment of human growth hormone deficiency. However, expression in inclusion bodies and low expression levels are enormous challenges for heterologous expression of hGH in Escherichia coli. OBJECTIVE: To increase the soluble expression of recombinant hGH with correct folding in E. coli. METHODS: We constructed a new recombinant expression plasmid containing the coding sequence of the outer membrane protein A (ompA3) which was used for the expression in Transetta (DE3) E. coli. In order to simplify the purification process and cleavage of recombinant proteins, the fusion sequence should contain hexahistidine-tag (His6) and enterokinase recognition sites (D4K). The effect of different expression conditions on recombinant hGH expression was optimized in flask cultivations. Furthermore, the periplasmic solution containing soluble hGH was purified by Ni-NTA affinity chromatography. Circular dichroism (CD), western blot and mass spectrometry analyses were used to characterize the protein. Moreover, the growth-promoting effect of the purified hGH was also evaluated by cell proliferation assay. RESULTS: High-level expression (800 µg/mL) was achieved by induction with 0.5 mM IPTG at 30°C for 10 hours. The purity of hGH was over 90%. The immunological activity, secondary structure and molecular weight of the purified hGH were consistent with native hGH. The purified hGH was found to promote the growth of MC3T3-E1 cells, and was found to show the highest activity at a concentration of 100 ng/mL. CONCLUSION: Our research provides a feasible and convenient method for the soluble expression of recombinant hGH in E. coli, and may lay a foundation for the production and application of hGH in the industry.