Preliminary study on preparation of cell-free system for protein expression

In the new era of “Omics”, the traditional techniques of protein expression in vivo can not come up with the exponential increase of genetic information. The cell-free protein synthesis system provides a new strategy of protein expression with advantages of rapid, convenient and high-throughput expression. The preparation of cell extracts, the optimization of substrate concentrations and the energy regeneration system are the key factors for the successful construction of cell-free protein expression system. In this work, the cell extract was prepared from RNase I- defective strain E. coli A19. The cell growth phase, the pressure for cell disruption and the storage condition of cell extracts were optimized. Meanwhile, the optimal substrate concentrations and the energy regeneration system were selected. Under the optimized conditions, the green fluorescent protein (GFP) reporter gene was expressed in the E. coli cell-free system with high expression level (Ca. 154 ?g/mL) which was 29 times higher than the expression level before optimization.     

High-expression of recombinant human concensus interferon-α by Pichia pastoris

The present work focused on the high expression of recombinant human consensus interferon-α (cIFN) by Pichia pastoris. The cycle of glycerol feeding, the strategy of methanol feeding and the optimum pH for protein induction were studied. The optimized strategies were a 4-h glycerol-feeding period, induction pH being kept at 5.0 and methanol concentration being kept under 5 g/L. The maximum dry cell weight, cIFN production and bioactivity obtained were 168, 1.24 g/L and 5.4 × 10⁷ U/mL, respectively. __________ Translated from Journal of East China University of Science and Technology (Natural Science Edition), 2007, 33(2): 172–176 [译自: 华东理工大学学报 (自然科学版)]     

Biotransformation of medium-chain alkanes using recombinant P450 monooxygenase from <Emphasis Type="Italic">Alcanivorax borkumensis</Emphasis> SK2 expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A bioconversion system for medium-chain alkanes was constructed by using a recombinant Escherichia coli whole-cell biocatalyst expressing P450 monooxygenase genes, ferredoxin, and ferredoxin reductase cloned from Alcanivorax borkumensis as an operon. The recombinant E. coli harboring the P450 gene and two related expression component enzymes, ferredoxin and ferredoxin reductase, was constructed in a single vector pET21(a) and successfully expressed in E. coli BL21(DE3) as a soluble form, showing a molecular weight of 53 kDa on 10% SDS-PAGE. When the cell-free extract of E. coli BL21 expressing p450 monooxygenase was subjected to reduced CO difference spectral analysis, a soret band near 450 nm appeared indicating that the cloned P450 was expressed as a functionally active enzyme. The E. coli cells harboring the expressed P450 gene were able to convert n-octane and 1-decene, producing approximately 450 μg/ml of n-octanol and 290 μg/ml of 1,2-epoxydecane, respectively, at pH 7.0 and 30 °C. However, the recombinant E. coli cells were not able to convert the branched alkane, 2,6,10,14-tetramethylpentadecane (C19).     

Recombinant aspartate aminotransferase-catalyzed synthesis of L-4-fluorophenylalanine

L-4-Fluorophenylalanine (FPhe) was prepared from 4-fluorophenylpyruvate (FPPA) catalyzed by aspartate aminotransferase (Asp-AT) of the recombinant E. coli BL21-pET/aspC. After 12 h enzymatic reaction, the FPPA conversion was over 90% and the yield of FPhe could be above 85% under the following optimal conditions: 37°C, pH value range of 5.0–8.0, 5.5 mass ratio of cell to FPPA, 0.6% (w/v) of Tween 80, 7.08 g/L FPPA, and 1.6 of molar ratio of L-Asp to FPPA. __________ Translated from Chemical Engineering (China), 2007, 35(10): 61–64 [译自: 化学工程]     

Efficient cell surface display of organophosphorous hydrolase using N-terminal domain of ice nucleation protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been used for detoxifying toxic organophosphate compounds. However, a whole cell biocatalyst system has an intrinsic problem due to substrate diffusion limitation by its cell membrane. As a strategy for reducing this diffusion barrier limitation to enhance whole cell biocatalytic activity, we engineered E. coli cells to target OPH on cell surface using ice nucleation protein (InaK) as a surface targeting motif, especially N-terminal domain of InaK (InaK-N). The whole cell OPH activities of the cells expressing InaK/OPH fusion constructs were higher (∼2.5-fold for InaK-N and ∼5.7-fold for combined N-and C-terminal domain of InaK (InaK-NC)) than that of the cells expressing cytosolic OPH. Interestingly, the membrane targeting efficiency of the cells expressing InaK-N/OPH fusion proteins was ∼2.2-fold higher compared to the cells expressing InaK-NC/OPH even though both whole cell and total cell lysate OPH activities were lower. Therefore, we found that the small size N-terminal domain of InaK is more efficient for targeting OPH on the cell surface, and the surface display of OPH using N-terminal InaK domain can reduce the mass-transfer problem in whole cell bioconversion system. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007     

Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems

Recombinant immunotoxins (RITs) are an effective class of agents for targeted therapy in cancer treatment. In this article, we demonstrate the straight-forward production and testing of an anti-CD7 RIT based on PE24 in a prokaryotic and a eukaryotic cell-free system. The prokaryotic cell-free system was derived from Escherichia coli BL21 Star^TM (DE3) cells transformed with a plasmid encoding the chaperones groEL/groES. The eukaryotic cell-free system was prepared from Chinese hamster ovary (CHO) cells that leave intact endoplasmic reticulum-derived microsomes in the cell-free reaction mix from which the RIT was extracted. The investigated RIT was built by fusing an anti-CD7 single-chain variable fragment (scFv) with the toxin domain PE24, a shortened variant of Pseudomonas Exotoxin A. The RIT was produced in both cell-free systems and tested for antigen binding against CD7 and cell killing on CD7-positive Jurkat, HSB-2, and ALL-SIL cells. CD7-positive cells were effectively killed by the anti-CD7 scFv-PE24 RIT with an IC₅₀ value of 15 pM to 40 pM for CHO and 42 pM to 156 pM for E. coli cell-free-produced RIT. CD7-negative Raji cells were unaffected by the RIT. Toxin and antibody domain alone did not show cytotoxic effects on either CD7-positive or CD7-negative cells. To our knowledge, this report describes the production of an active RIT in E. coli and CHO cell-free systems for the first time. We provide the proof-of-concept that cell-free protein synthesis allows for on-demand testing of antibody–toxin conjugate activity in a time-efficient workflow without cell lysis or purification required.     

Expression of redesigned mussel silk-like protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Silks have been used widely for human beings due to their several extraordinary properties. Until now, the studies on silk proteins have mainly focused on spiders and silkworms. Because silk properties are organism-dependent, novel silk protein types can be found and developed through investigation of new silk-bearing organisms. We noticed that marine mussel has silk-like domains containing many repeats with abundance of glycine and alanine. In the present work, we redesigned mussel-derived silk-like gene sequence which contains alternating repeated and nonrepeated regions with optimized codons for Escherichia coli. For successful expression of recombinant mussel silk-like protein in E. coli cells, we employed several experimental strategies, including use of strong promoter, cold shock expression, and genetic fusions. We observed significant repression on cell growths by even low expression levels of soluble mussel silk-like proteins in cold shock- and glutathione s-transferase (GST) fusion-based systems. Thus, we finally used baculoviral polyhedrin protein as a fusion partner and successfully expressed insoluble mussel silk-like protein with relatively high expression level and without cell growth repression in E. coli.     

Modeling of cell growth andphoA-directed expression of cloned genes in recombinantEscherichia coli

A mathematical model was formulated to describe growth and cloned protein production in the recombinantEscherichia coli cells containingphoA-directed expression systems. Kinetic parameters for the strains with two fusion genes (phoA- lacZ either on the chromosome or on a multicopy plasmid andphoA- amyE on a multicopy plasmid) were estimated and compared to analyze the effects of cloning site (chromosome and plasmid), product type(E. coli β-galactosidase andBacillus subtilis α-amylase), and culture temperature on the cell’s behavior. The presence of a multicopy plasmid reduced the specific growth rate and the phosphate uptake rate of the cell, both by 10%, compared with those of the chromosome-integrated strain. The overexpression ofB. subtilis a-amylase decreased the specific growth rate and the glucose consumption rate more than the β-galactosidase overproduction system. The presence of multiple copies of thephoA promoter on either an intactphoA gene or the fusion gene reduced both the repression and derepression efficiencies. Culture temperatures showed a significant effect on a-amylase production. A temperature of 30°C is more desirable than 37°C for α-amylase production in the recombinantE. coli containing thephoA promoter.     

Studies on culture condition of new marine bacterium Zooshikella sp. SY01

New marine bacterium Zooshikella sp. SY01, producer of prodigiosin, was isolated from the seawaters of Sanya Bay. The culture conditions of this bacterium were investigated. Zooshikella sp. SY01 was cultured in 2216E media which contained tryptophan, histidine, lactonic acid, camphor, limonene, casein, diphenyl guanidine, coumarin and 1,3-dinitrobenzene, respectively. After 5 days cultivation, the extracts of different culture broths were detected by direct infusion mass spectroscopy using positive ESI mode. As the results, tryptophan, histidine and casein didn’t show any observable influences on the biosynthesis of prodigiosin. Lactonic acid, camphor, limonene, diphenyl guanidine, coumarin could inhibit the bacterium growth and prodigiosin biosynthesis to a certain extent, slower the culture broth to turn red. However, 1,3-dinitrobenzene inhibited the bacteria to produce prodigiosin completely. MS data suggested that various metabolites with chemodiversity were produced in different culture media. In particular, a series of high-molecular-weight compounds with high relative abundances were observed in the medium containing limonene. To further optimize the culture condition, more new prodigiosin analogues and lead compounds can be obtained and the goal of “one strain-many compounds” can be achieved. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2007, 46(6): 55–58 [译自: 中山大学学报(自然科学版)]     

A Clickable Photosystem I,Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration**

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b₆f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP⁺ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.     

Multiple-copy-gene integration on chromosome of <Emphasis Type="Italic">Escherichia coli</Emphasis> for beta-galactosidase production

Recombinant E. coli strains with 1–3 copies of lacZ genes on their chromosomes were constructed and their β-galactosidase (β-gal) expressions were examined. Serial dilution cultures were used to analyze the long-term genetic stability of the recombinant lacZ genes of the chromosomal or plasmid expression system. The strain with a 3-copy lacZ on the chromosome has a sustainable β-gal expression through 60 hours. However, the β-gal activity of the plasmid expression system lasted less than 36 hours under a no selection condition. Obviously, the genetic stability of the chromosomal expression system demonstrated in this study is better than that of the plasmid expression system under nonselective condition, such as a medium without antibiotics. The results demonstrated that the strains with a multiple-copy-gene on the chromosome are useful for protein production in industrial repeated fed-batch fermentation. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

Effect of yeast extract on the expression of thioredoxin–human parathyroid hormone from recombinant Escherichia coli

Terrific broth, a complex medium containing a high content of yeast extract, was chosen to cultivate recombinant Escherichia coli with the plasmid encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH). The volumetric yield of Trx–hPTH fusion protein in the culture with Terrific broth reached about 800 mg L^?1 after optimization. It was found that high content of yeast extract in Terrific broth promoted cell growth and Trx–hPTH fusion protein production. However, the more interesting fact was confirmed that high content of yeast extract was also responsible for the high‐level expression of Trx–hPTH fusion protein without specific inducer addition. Further studies indicated that the expression levels of Trx–hPTH fusion protein without specific inducer addition varied greatly with the content and the source of yeast extract contained in the media. Considering that some proteins are toxic to the host and their continuous expression may result in decreasing plasmid stability and protein yields, one should be cautious in selecting yeast extract in media for cultivating E. coli with plasmids carrying toxic genes under T7 control. Copyright © 2006 Society of Chemical Industry     

Study of simultaneous saccharification and fermentation for steam exploded wheat straw to ethanol

Although simultaneous saccharification and fermentation (SSF) has been investigated extensively, the optimum condition for SSF of wheat straw has not yet been determined. Dilute sulfuric acid impregnated and steam explosion pretreated wheat straw was used as a substrate for the production of ethanol by SSF through orthogonal experiment design in this study. Cellulase mixture (Celluclast 1.5 l and β-glucosidase Novozym 188) were adopted in combination with the yeast Saccharomyces cerevisiae AS2.1. The effects of reaction temperature, substrate concentration, initial fermentation liquid pH value and enzyme loading were evaluated and the SSF conditions were optimized. The ranking, from high to low, of influential extent of the SSF affecting factors to ethanol concentration and yield was substrate concentration, enzyme loading, initial fermentation liquid pH value and reaction temperature, respectively. The optimal SSF conditions were: reaction temperature, 35°C; substrate concentration, 100 g·L⁻¹; initial fermentation liquid pH, 5.0; enzyme loading, 30 FPU·g⁻¹. Under these conditions, the ethanol concentration increased with reaction time, and after 72 h, ethanol was obtained in 65.8% yield with a concentration of 22.7 g·L⁻¹. __________ Translated from Chemical Engineering (China), 2007, 35(12): 42–45 [译自: 化学工程]     

A fermentation strategy for production of recombinant protein subjected to plasmid instability

The appearance of plasmid-losing cells in a recombinant Escherichia coli culture was observed when the cell mass became doubled after induction, which corresponded to the timing of cell fission. Accordingly, a two-stage fermentation strategy capable of maintaining plasmid stability without selective pressure in a recombinant E. coli culture was proposed. In the first stage (cell growth stage), a high cell density culture was obtained by incubating the cells in the R medium. In the second stage (producing stage), the cells were devoted to producing the recombinant protein by introducing the fresh LB medium supplemented with isopropyl-β-d-thiogalactopyranoside (IPTG). It was necessary to prevent the doubling in the cell mass after induction; otherwise cell fission would occur and generate plasmid-losing cells. The present strategy is expected to be extensively applicable in recombinant E. coli cultures. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007.     

Trifluoroselenomethionine: A New Unnatural Amino Acid

Trifluoroselenomethionine (TFSeM), a new unnatural amino acid, was synthesized in seven steps from N‐(tert‐butoxycarbonyl)‐l ‐aspartic acid tert‐butyl ester. TFSeM shows enhanced methioninase‐induced cytotoxicity, relative to selenomethionine (SeM), toward HCT‐116 cells derived from human colon cancer. Mechanistic explanations for this enhanced activity are computationally and experimentally examined. Comparison of TFSeM and SeM by selenium EXAFS and DFT calculations showed them to be spectroscopically and structurally very similar. Nonetheless, when two different variants of the protein GB1 were expressed in an Escherichia coli methionine auxotroph cell line in the presence of TFSeM and methionine (Met) in a 9:1 molar ratio, it was found that, surprisingly, 85 % of the proteins contained SeM residues, even though no SeM had been added, thus implying loss of the trifluoromethyl group from TFSeM. The transformation of TFSeM into SeM is enzymatically catalyzed by E. coli extracts, but TFSeM is not a substrate of E. coli methionine adenosyltransferase.     

Parallel Bubble Columns with Fed-Batch Technique for Microbial Process Development on a Small Scale

12 small-scale bubble columns, each and every column with a total volume of 500 ml, individually controlled distribution of sterile and humidified air and a pH-probe, were operated in an incubation chamber with temperature control. Intermittent feeding of substrate or inducer, as well as of base for parallel pH-control was achieved by connecting a precision syringe pump to a valve distributor with one 2/2-way miniature valve for each bubble column. This new parallel bio-reactor technique was applied for optimization of the feed profile of the chemical inducer isopropyl-β-D-thiogalactoside (IPTG) to improve the expression of a foreign protein (guanosin-5′-diphosphate-α-D-mannose-pyrophosphorylase, GDP-manPP) under the control of the lac promoter in Escherichia coli. A mean cell density of 8 g l^–1 of recombinant E. coli was achieved in parallel fed-batch fermentations within 9.5 h due to a sufficient oxygen transfer (k_La of up to 0.2 s^–1) and parallel pH-control. The GDP-manPP activity was improved by more than 100 % compared to the standard IPTG pulse within 3 sets of parallel fed-batch fermentations. A Genetic Algorithm was used for optimization of the IPTG feed profile. Scale-up of the optimized fed-batch process into the stirred tank reactor (scale-up factor of 20) was possible, if all reaction conditions were copied exactly with respect to the reactor volume. A final GDP-manPP activity of 715 U l^–1 was measured 3 h after IPTG induction was finished.     

Kinetic Analysis of Terminal and Unactivated C?H Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase‐Based Whole‐Cell Biocatalysis

The alkane monooxygenase AlkBGT from Pseudomonas putida GPo1 constitutes a versatile enzyme system for the ω‐oxyfunctionalization of medium chain‐length alkanes. In this study, recombinant Escherichia coli W3110 expressing alkBGT was investigated as whole‐cell catalyst for the regioselective biooxidation of fatty acid methyl esters to terminal alcohols. The ω‐functionalized products are of general economic interest, serving as building blocks for polymer synthesis. The whole‐cell catalysts proved to functionalize fatty acid methyl esters with a medium length alkyl chain specifically at the ω‐position. The highest specific hydroxylation activity of 104 U g_CDW⁻¹ was obtained with nonanoic acid methyl ester as substrate using resting cells of E. coli W3110 (pBT10). In an optimized set‐up, maximal 9‐hydroxynonanoic acid methyl ester yields of 95% were achieved. For this specific substrate, apparent whole‐cell kinetic parameters were determined with a V_max of 204±9 U g_CDW⁻¹, a substrate uptake constant (K_S) of 142±17 μM, and a specificity constant V_max/K_S of 1.4 U g_CDW⁻¹ μM ⁻¹ for the formation of the terminal alcohol. The same E. coli strain carrying additional alk genes showed a different substrate selectivity. A comparison of biocatalysis with whole cells and enriched enzyme preparations showed that both substrate availability and enzyme specificity control the efficiency of the whole‐cell bioconversion of the longer and more hydrophobic substrate dodecanoic acid methyl ester. The efficient coupling of redox cofactor oxidation and product formation, as determined in vitro, combined with the high in vivo activities make E. coli W3110 (pBT10) a promising biocatalyst for the preparative synthesis of terminally functionalized fatty acid methyl esters.     

Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing

Bacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial bacteriocins are alternatives to conventional antibiotics and are characterized by unique cell-killing mechanisms, including pore formation on cell membranes, nuclease activity, and cell wall synthesis inhibition. Here, we used cell-free protein synthesis to rapidly evaluate the anti-biofilm activities of colicins E1, E2, and E3. We found that E2 (with DNase activity) most effectively killed target biofilm cells (i.e., the K361 strain) while leaving nontargeted biofilms intact. We then engineered probiotic Escherichia coli microorganisms with genetic circuits to controllably synthesize and secrete colicin E2, which successfully inhibited biofilms and killed preformed indicator biofilms. Our findings suggest that colicins rapidly and selectively kill target biofilm cells in multispecies biofilms and demonstrate the potential of using microorganisms engineered to produce antimicrobial colicin proteins as live therapeutic strategies to treat biofilm-associated infections.