植物中的冷激蛋白

植物冷激蛋白结构保守,具有RNA结合位点(S1结构域)或类似的β折叠构成的桶状结构,它与原核生物冷激蛋白同源性高,通过转录、翻译等的调节完成其生理功能。     

无细胞蛋白合成体系实现胰岛素原可溶性表达

胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。     

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell-free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are “cell-free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.     

Development of an E. coli strain for cell-free ADC manufacturing

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the Escherichia coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with nonnatural amino acids incorporated at specific locations for producing homogeneous antibody–drug conjugates (ADCs). While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for nonnatural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a 5-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing nonnatural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with the batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.     

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Exploiting Leishmania tarentolae cell-free extracts for the synthesis of human solute carriers

Abstract

Cell-free protein production offers a versatile alternative to complement in vivo expression systems. However, usage of prokaryotic cell-free systems often leads to non-functional proteins. We modified a previously designed cell-free system based on the protozoan Leishmania tarentolae, a parasite of the Moorish gecko Tarentola mauritanica, together with a species-independent translational sequences-based plasmid to produce human membrane proteins in 2 hours reaction time. We successfully established all four commonly used expression modes for cell-free synthesis of membrane proteins with a human organic anion transporter, SLC17A3, as a model membrane protein: (i) As precipitates without the addition of any hydrophobic environment, (ii) in the presence of detergents, (iii) with the addition of liposomes, and (iv) supplemented with nanodiscs. We utilized this adapted system to synthesize 22 human solute carriers from 20 different families. Our results demonstrate the capability of the Leishmania tarentolae cell-free system for the production of a huge variety of human solute carriers in the precipitate mode. Furthermore, purified SLC17A3 shows the formation of an oligomeric state.     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

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.     

Membrane- and cell wall-associated heat shock proteins in two genotypes of barley seedlings

ABSTRACT

The heat-shock (HS) response of two genotypes (cv. Onice, winter type, and cv. Georgie, spring type) of barley (Hordeum vulgare L.) was compared. Protein synthesis was markedly reduced by HS in roots and coleoptiles of both genotypes. The reduction in cv. Onice was higher than in cv. Georgie. The pattern of cytosolic, membrane and cell wall HSPs was analysed by SDS-PAGE in coleoptiles and roots of the two genotypes. Differences and similarities in coleoptiles and roots of the same genotype and between the two genotypes were observed. In roots of the genotype Onice, LMW and HMW HSPs isolated from the cytosol, membranes and cell walls were resolved into a diverse array of polypeptides by two-dimensional gel electrophoresis. Present results confirm the de novo synthesis of cytosolic and membrane HSPs, and demonstrate, for the first time, their presence in cell walls.     

A rational approach to improving titer in Escherichia coli-based cell-free protein synthesis reactions

Cell-free protein synthesis (CFPS) is an established method for rapid recombinant protein production. Advantages like short synthesis times and an open reaction environment make CFPS a desirable platform for new and difficult-to-express products. Most recently, interest has grown in using the technology to make larger amounts of material. This has been driven through a variety of reasons from making site specific antibody drug conjugates, to emergency response, to the safe manufacture of toxic biological products. We therefore need robust methods to determine the appropriate reaction conditions for product expression in CFPS. Here we propose a process development strategy for Escherichia coli lysate-based CFPS reactions that can be completed in as little as 48 hr. We observed the most dramatic increases in titer were due to the E. coli strain for the cell extract. Therefore, we recommend identifying a high-producing cell extract for the product of interest as a first step. Next, we manipulated the plasmid concentration, amount of extract, temperature, concentrated reaction mix pH levels, and length of reaction. The influence of these process parameters on titer was evaluated through multivariate data analysis. The process parameters with the highest impact on titer were subsequently included in a design of experiments to determine the conditions that increased titer the most in the design space. This proposed process development strategy resulted in superfolder green fluorescent protein titers of 0.686 g/L, a 38% improvement on the standard operating conditions, and hepatitis B core antigen titers of 0.386 g/L, a 190% improvement.     

Cold shock response inLactococcus lactis ssp.diacetylactis

The acquired freeze-thaw tolerance was investigated forLactococcus lactis ssp.diacetylactis. Pretreatment of microorganisms at less severe temperatures to initiate cold tolerance gaveL. lactis ssp.diacetylactis improved cell viability after successive freezings and thawings. The ability of cells to survive freeze-thaw was dependent on factors experienced prior to freezing. Factors affecting lactic acid bacteria survival during freeze-thaw cycles were found to be different diluents, growth phase, and different cold temperatures. Viability experiments showed that this strain displaying cold shock cryotolerance had an improved survival capacity in stationary phase. The plasmid contents of lactic acid bacteria isolated from different types, DRC-2 and DRC-2C, were examined and compared with the plasmid contents of culture collection strains both before and after cold shock treatment. Using agarose gel electrophoresis, no obvious correlation between the cold shock response and the number of plasmids in the cell could be observed.     

Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis

Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.     

High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids

We describe an E. coli-based cell-free system for the production of proteins with a non-natural amino acid (nnAA) incorporated site-specifically (modified protein). The mutant Methanococcus jannaschii tyrosyl-tRNA synthetase (mTyrRS) and tRNA(Tyr) pair were used as orthogonal elements. The mTyrRS experienced proteolysis and modified protein yields improved with higher synthetase addition (200-300 microg/mL). Product yields were also improved by increasing levels of total protein to 20 mg protein/mL and available vesicle surface area to 0.5 m(2)/mL. This new E. coli-based cell-free procedure produced up to 400 microg/mL of eCAT109pAz, 660 microg/mL of eDHFR10pAz, and 210 microg/mL of mDHFR31pAz with p-azido-L-phenylalanine (pAz) incorporated site-specifically at the amber nonsense codon. O-methyl-L-tyrosine and p-acetyl-L-phenylalanine were incorporated by similar protocols. The desired specificity for incorporation of the nnAA by the cell-free system was confirmed. Additionally, the modified proteins were enzymatically active and reactive for copper(I)-catalyzed (3 + 2) cycloadditions (click chemistry).     

Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli

The utilization efficiency of a secondary energy source in a cell-free protein synthesis system can be improved by use of a metabolic inhibitor. Oxalate, a potent inhibitor of phophoenolpyruvate synthetase, substantially increased the yield of chloramphenicol acetyltransferase synthesis through the enhanced supply of ATP. Oxalate, at 2.7 mM, increased the synthesis yield by 47% when successive amino acids additions prevent amino acid depletion during protein synthesis. These results suggest that cell-free protein synthesis efficiency could also be improved by disrupting the gene encoding phosphoenolpyruvate synthetase.     

Acid shock proteins of Escherichia coli

Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell-free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.     

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.