Production Of Homologous Proteins Research Articles

Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

The pyruvate:ferredoxin oxidoreductase of the thermophilic acetogen, Thermoanaerobacter kivui.

Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low‐potential ferredoxin (Fd2−) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg−1) and ferredoxin (Fd; 27.2 U·mg−1) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein−1, consistent with the presence of three predicted [4Fe–4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His‐tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron‐bifurcating enzymes, to keep a constant level of reduced Fd.

Expression Of Human Basic Fibroblast Growth Factor Mediated By Mini Intein In Bacillus Subtilis

Bacillus subtilis (B. subtilis) is an ideal host system in production of homologous proteins. However, the production of heterologous proteins in B. subtilis is rare due to low expression levels encountered in most cases. Inteins, also known as ‘protein intron’, which is capable of excised itself from its fusion partners, have been employed for the expression of recombinant proteins in various host systems especially in Escherichia coli (E. coli) but yet, only few paucity of employment of inteins for protein expression in B. subtilis has been found. In this communication, we demonstrated that B. subtilis was able to facilitate auto-cleavages between intein and C-extein. The construct, pECBS1-H6-DnaE-bFGF, in which a 6x His-tag (H6) and basic fibroblast growth factor (bFGF) were fused at the N- and C-terminus of Asp DnaE (intein) respectively, was shown to be capable of processing intracellular expression and auto-cleavages of bFGF with same primary sequence as Homo Sapiens. Moreover, switching shake of flask cultivation to small fermentative scale yielded 113 mg L-1 of biologically active bFGF. This approach of using intein Asp DnaE for the production of heterologous proteins is highly productive and should be explored further for industrial application.

Endoplasmic reticulum stress induced by lipopolysaccharide is involved in the association between inflammation and autophagy in INS‑1 cells.

Type 2 diabetes is a chronic inflammatory disease. Autophagy, the dynamic process of lysosomal degradation of damaged organelles and proteins, may protect β-cells from destruction by inflammation in type 2 diabetes. The present study investigated the role of autophagy, inflammation and endoplasmic reticulum (ER) stress in type 2 diabetes. INS-1 cells were incubated with lipopolysaccharide. The chemical chaperone 4-phenylbutyric acid was used to inhibit ER stress, and 3-methyadenine (3-MA) was used to inhibit autophagy. Apoptosis was detected by flow cytometry and cell proliferation using Cell Counting kit-8 solution. Light chain-3B, interleukin (IL) 1β, caspase-1 and C/EBP homologous protein production were assessed by western blotting, and rat activating transcription factor 4 and rat binding immunoglobulin heavy chain protein gene expression were determined by real-time reverse transcription-polymerase chain reaction. The results showed that inhibiting autophagy with 3-MA unexpectedly contributed to cell death in β-cells. This response was associated with an increase in inflammatory cytokines, including IL1β and caspase-1. Inhibiting ER stress with 4-phenylbutyric acid led to a decrease in cell apoptosis. These results showed that autophagy may have a protective effect by reducing inflammatory cytokines in β-cells. In addition, the inositol-requiring enzyme 1 pathway mediated the ER stress associated with autophagy and inflammatory cytokines (IL1β and caspase-1). Therefore, inflammatory cytokines may be critical signalling nodes, which are associated with ER stress-mediated β-cell death.

New Promoters to Improve Heterologous Protein Production in Aspergillus vadensis

Aspergillus is a widely used host organism for the industrial production of homologous and heterologous proteins. Although Aspergillus niger is most commonly used, a close relative of this species, Aspergillus vadensis, has been suggested as a suitable and more favourable alternative due in part, to the low levels of extracellular proteases which it produces. Despite much progress being made in the hyper production of homologous proteins, the yields obtained for heterologous proteins have still not reached a comparable level. Genetic strategies, including the development of strong constitutional promoters have been shown to lead to an increase in the levels of recombinant protein production. In this study, six novel constitutional promoters from A. niger (pef1α, ptktA, pef1β, ptal1, pcetA and ppgkA) and a further five from A. vadensis (pef1α, prps31, pgpdA, pubi1 and poliC) were tested in A. vadensis using a gene encoding a secreted arabinofuranosidase from Fusarium oxysporum as a reporter for heterologous protein production. Remarkably, 9 of the 11 promoter constructs tested resulted in higher ABF activity than the commonly used gpdA promoter. While this could partly be assigned to the number of copies of the expression cassette in the transformants, clear differences in productivity of the promoters could be observed.

Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5' and 3' ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970U/mg protein and 12.3mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.

Recent Studies of Protein Secretion by Filamentous Fungi.

Filamentous fungi have been widely exploited for the homologous and heterologous protein production, because of the high capacity of their protein secretion machinery. However, the production of heterologous proteins is often limited while the production of homologous proteins can be very high. Various researches have reported the methods for overcoming this problem and some techniques, such as the fusion gene system, improve the production of heterologous proteins. Recently, the molecular biological study of solid-state culture attracts the attention, because the long history of biological studies has shown that the productivity of protein in the solid-state culture frequently exceeds the productivity of protein in the submerged culture. The recent researches of solid-state culture have revealed the new aspects of protein production in filamentous fungi. Solid-state specific gene expression was observed in the glaB and pepA genes of Aspergillus oryzae . A GC-box and HSE element of the glaB promoter region affected solid-state specific gene expression of this gene. Solid-state culture-specific release of enzymes from the cell wall was also observed in the production of β-glucosidases in Aspergillus kawachii . Extracellular soluble polysaccharide (ESP) from A. kawachii was concerned with the location of β-glucosidases. Moreover, ESP and the cell wall fraction of A. kawachii were shown to be involved in the stability of β-glucosidases. The knowledge of the molecular biology of solid-state culture should provide new approaches for the production of both homologous and heterologous proteins in both submerged culture and solid-state culture of filamentous fungi.