Extracellular Production Of Recombinant Proteins Research Articles

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Protein Expression and Purification of Romiplostim and Analysis of Its Secretory Production Using an In Silico Investigated Signal Peptide in E. Coli.

Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in Escherichia coli. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in E. coli by using signal peptide sequences due to its advantages compared to intracellular production. In this study, the effect of the pelB signal peptide on Romiplostim production was analyzed. The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 program, and expression, purification, and biological activity of the recombinant protein were analyzed. In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group. Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.

Enhancing extracellular production of recombinant proteins in Escherichia coli by co-expressing with a haloarchaeal protein containing a putative LolA-like domain.

Escherichia coli represents one of the most widely used hosts for recombinant protein production, but its limited capacity for producing extracellular proteins is often cited as a drawback. NJ7G_0991 is an extracellular protein of the haloarchaeon Natrinema sp. J7-2 and comprises a signal peptide, a putative LolA-like domain, and a C-terminal domain of unknown function. Here, we found that the full-length (0991) and the C-terminal domain-deletion variant (0991ΔC) of NJ7G_0991, but not its signal peptide-deletion variant (0991ΔS), were efficiently released into the culture supernatant of E. coli without extensive cell lysis as determined by β-galactosidase activity assay. After lysozyme treatment, E. coli cells producing 0991 or 0991ΔC, but not 0991ΔS, were converted from rod-shaped forms to spheres, suggesting that the secretion of 0991 or 0991ΔC into the periplasm leads to an increase of outer membrane permeability of E. coli. A pelB signal peptide was fused to the N-terminus of the LolA-like domain, and the resulting variant PelB-0991ΔC could be released into the culture supernatant of E. coli more efficiently than 0991ΔC. By using PelB-0991ΔC as a co-expression partner, the extracellular production level of a recombinant thermostable subtilase WF146 could be enhanced by up to 14-fold, and the extracellular concentration of an active site variant of WF146 (WF146-SA) reached up to 129 mg/l. To the best of our knowledge, this is the first report on archaeal protein-based co-expression system for extracellular production of recombinant proteins in E. coli. KEY POINTS: • The haloarchaeal protein NJ7G_0991 can be efficiently released into the culture supernatant of E. coli. • The recombinant NJ7G_0991 increases the outer membrane permeability of E. coli. • The LolA-like domain of NJ7G_0991 can be used as a co-expression partner to improve extracellular production of recombinant proteins in E. coli.

Characterization and enhanced extracellular overexpression of a new salt-activated alginate lyase.

Alginate lyases (EC 4.4.2.3/4.4.2.11) have been applied to produce alginate oligosaccharides, which have physiological advantages such as prebiotic and antidiabetic effects, and are of benefit in the food and pharmaceutical industries. Extracellular production of recombinant proteins in Escherichia coli presents advantages including simplified downstream processing and high productivity; however, the presence of certain signal peptides does not always ensure successful secretion, which make the extracellular production of alginate lyase in E. coli rarely reported but of great significance. A PL7 family alginate lyase, Aly01, with its native signal peptide from Vibrio natriegens SK42.001, was identified, characterized, and extracellularly expressed in E. coli. The enzyme specifically released trisaccharide from alginate and was strictly NaCl activated. Green fluorescent protein (GFP) was fused with the Aly01 signal peptide and successfully secreted in E. coli to expand the feasibility of using this signal peptide to produce other heterologous proteins extracellularly. Through a synergistic strategy of utilizing Terrific Broth (TB) medium supplemented with 120 mmol L-1 glycine and 10 mmol L-1 calcium, the lag phase of protein secretion was reduced to 3 h from 12 h; meanwhile calcium remedied glycine-related cell growth impairment, leading to further enhancement of overall enzyme productivity, reaching a maximum of 4.55 U mL-1 . A new salt-activated alginate lyase, Aly01, was identified and characterized. E. coli employed its signal peptide and extracellularly expressed both Aly01 and a GFP, which indicated the signal peptide of Aly01 could be a powerful tool for extracellular production of other heterologous proteins in E. coli. © 2021 Society of Chemical Industry.

Inhibition of E. coli Host RNA Polymerase Allows Efficient Extracellular Recombinant Protein Production by Enhancing Outer Membrane Leakiness.

With the growing interest in continuous cultivation of Escherichia coli, secretion of product to the medium is not only a benefit, but a necessity in future bioprocessing. In this study, it is shown that induced decoupling of growth and heterologous gene expression in the E. coli X-press strain (derived from BL21(DE3)) facilitates extracellular recombinant protein production. The effect of the process parameters temperature and specific glucose consumption rate (qS ) on growth, productivity, lysis and leakiness, is investigated, to find the parameter space allowing extracellular protein production. Two model proteins are used, Protein A (SpA) and a heavy-chain single-domain antibody (VHH), and performance is compared to the industrial standard strain BL21(DE3). It is shown that inducible growth repression in the X-press strain greatly mitigates the effect of metabolic burden under different process conditions. Furthermore, temperature and qS are used to control productivity and leakiness. In the X-press strain, extracellular SpA and VHH titer reach up to 349 and 19.6mg g-1 , respectively, comprising up to 90% of the total soluble product, while keeping cell lysis at a minimum. The findings demonstrate that the X-press strain constitutes a valuable host for extracellular production of recombinant protein with E. coli.

Isolation of a Novel Bacterial Strain Capable of Producing Abundant Extracellular Membrane Vesicles Carrying a Single Major Cargo Protein and Analysis of Its Transport Mechanism.

Extracellular membrane vesicles (EMVs) play an important role in various bacterial activities. EMVs have potential for use as vaccines, drug-delivery vehicles, platforms for extracellular production of recombinant proteins, and so on. In this study, we newly isolated a cold-adapted bacterium, Shewanella vesiculosa HM13, which abundantly produces EMVs, characterized them, and analyzed their cargo transport mechanism. S. vesiculosa HM13, isolated from the intestine of a horse mackerel as a prospective host for a low-temperature secretory protein expression system, produced a single major secretory protein, P49, of unknown function in the culture supernatant. Analysis using sucrose density gradient ultracentrifugation indicated that P49 is a cargo protein carried by EMVs. S. vesiculosa HM13 displayed extensive blebbing on the surface of the outer membrane, and the size of blebs was comparable to that of EMVs. These blebs are thought to be precursors of the EMVs. Disruption of the P49 gene resulted in only a marginal decrease in the EMV production, indicating that the EMVs are produced even in the absence of the major cargo protein. Whole genome sequencing of S. vesiculosa HM13 revealed that this bacterium has a gene cluster coding for a non-canonical type II protein secretion system (T2SS) homolog in addition to a gene cluster coding for canonical T2SS. The P49 gene was located downstream of the former gene cluster. To examine the role of the putative non-canonical T2SS-like translocon, we disrupted the gene coding for a putative outer membrane channel of the translocon, named GspD2. The gspD2 disruption lead to disappearance of P49 in the EMV fraction, whereas the production of EMVs was not significantly affected by this mutation. These results are indicative that the T2SS-like machinery functions as a novel type of protein translocon responsible for selective cargo loading to the EMVs. We also found that GFP fused to the C-terminus of P49 expressed in S. vesiculosa HM13 was transported to EMVs, indicating that P49 is useful as a carrier to deliver the fusion partner to EMVs. These findings deepen our understanding of the mechanism of biogenesis of EMVs and facilitate their applications.

Cloning and expression of pullulanase from Bacillus subtilis BK07 and PY22 in Pichia pastoris

In this study, pullulanase genes from a wild isolate B. subtilis BK07 and B. subtilis PY22 (mutant strain derived from B. subtilis 168) were transformed into P. pastoris KM71H. Extracellular recombinant protein production was achieved with methanol induction under the regulation of AOX1 promoter utilizing the Saccharomyces cerevisiae α-mating factor sequence for extracellular secretion. The molecular weight of the recombinant enzymes BK07pul and PY22pul were both approximately 90 kDa. Both enzymes showed highest activity at 40 °C, however PY22pul showed optimum activity at pH 6 whereas, BK07pul had highest activity at pH 8. BK07pul and PY22pul activities were determined as 8.46 U/mL and 15 U/mL. The enzyme stability of BK07pul was higher (89%) than PY22pul (68%) where relative activity was determined as activity remaining after 1 h at corresponding optimum conditions for each. Amino acid homology evaluation revealed the two enzymes had 80% identity in primary structure. The presence of conserved sequences consisting of 7 amino acids (YNWGYDP) in both enzymes confirmed these to be type I pullulanases, capable of hydrolyzing α-1,6 glucosidic bonds of pullulan resulting in maltotriose units.

Deleting mrdA and mrcB to significantly improve extracellular recombinant protein production in Escherichia coli

In this work, two key genes (mrdA and mrcB) involved in cell wall biosynthesis were deleted (singly and doubly) in Escherichia coli, and the effects on extracellular recombinant protein production were investigated. The mrdA and mrcB genes encode penicillin-binding protein (PBP) 2 and PBP1B, respectively. The growth of deletion mutants was not significantly inhibited compared with control cells, but cell morphology was altered. The concentration of intracellular soluble peptidoglycan was increased in deletion mutants, especially the double deletion mutant. Extracellular protein production was significantly improved in deletion mutants compared with controls, in the order double deletion > single deletion of mrcB > single deletion of mrdA. Extracellular green fluorescent protein production by the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-gfp was most significantly enhanced compared with control cells (2.6-fold). Extracellular production of recombinant fibroblast growth factor receptor 2 and collagen E4 was also improved in deletion mutants compared with control cells. Extracellular amylase activity of the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased 5.2-fold relative to control cells, but activity in BL21 ΔmrdA::pET28a-amyk and BL21 ΔmrcB::pET28a-amyk single deletion mutants was only increased 1.7- and 4.3-fold, respectively. Extracellular α-galactosidase activity of deletion mutants was also improved, especially the double deletion mutant (2.6-fold). Membrane permeability of deletion mutants was improved compared with control cells, which might increase extracellular recombinant protein production.

Genetic engineering modification and fermentation optimization for extracellular production of recombinant proteins using Escherichia coli.

As a common expression host, Escherichia coli has received more and more attention due to the recently developed secretory expression system, which offers advantages like reduced downstream bioprocesses and improved product quality. These advantages, coupled with high-density fermentation technology, make it a preferred system for large-scale production of many proteins utilized in industry and agriculture at a reduced process cost. To improve the secretion efficiency of target proteins, various strategies, including signal peptide optimization, periplasmic leakage, and chaperones co-expression have been developed. In addition, the optimization of the fermentation conditions such as temperature, inducer, and medium were also taken into account for the extracellular production in the high-density fermentation to reduce the cost of production. Here, these strategies ranging from genetic engineering to fermentation optimization were summarized for the future guidance of extracellular production of recombinant proteins using E. coli.

Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C

BackgroundOur laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins.ResultsWhen B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process.ConclusionsThis is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

Endogenous signal peptides in recombinant protein production by Pichia pastoris: From in-silico analysis to fermentation

For extracellular recombinant protein production, the efficiency of five endogenous secretion signal peptides (SPs) of Pichia pastoris, SP13 (MLSTILNIFILLLFIQASLQ), SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), SP34 (MRPVLSLLLLLASSVLA), selected based on their D-score which quantifies the signal peptide-ness of a given sequence segment, was investigated using recombinant human growth hormone (rhGH) as the model protein. The expression was conducted under glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). The highest secretion efficiency among endogenous SPs was obtained by SP23 followed by SP24, SP34, SP13 and SP26, respectively. The fermentation characteristics of rhGH production by the use of SP23, the most favorable endogenous SP of P.pastoris, and Saccharomyces cerevisiae α-mating factor prepro sequence (α-MF) were compared. With respect to the SP23 which is 73 amino acids shorter in length compared to α-MF, in high cell density cultures, where carbon and energy source are limited, the substitution of SP23 for α-MF seems promising. α-MF higher secretion efficiency was verified by major physicochemical properties including hydropathy index, isoelectric point, and aliphatic index. Regarding the examined endogenous SPs, there was no clear correlation between secretion efficiency and major physicochemical properties when each of these properties was considered independently. To find a correlation, factors such as protein N-terminus effect, length of the SP, secondary structure of the SP, and interactions of the selected properties should also be investigated.

An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100

Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ–AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ–AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ–AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ–AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the β-galactosidase activity test showed that the increased extracellular rZZ–AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.

Suppression of Bm-Caspase-1 Expression in BmN Cells Enhances Recombinant Protein Production in a Baculovirus Expression Vector System

Baculovirus expression vector systems (BEVSs) have been widely used for recombinant protein production. Many studies have tried to improve the recombinant protein production by either modification of BEVSs vectors or by cell line selection. In this study, using a modified shRNA vector, we established a stable Bombyx mori cell line that significantly inhibits expression of caspase-1 after selection. We further compared cell proliferation and viability between caspase-1-suppressed cells and control cells, and found that there is no significant difference in these stable cell lines. We utilized these cell lines to analyze recombinant protein production after infection with recombinant baculovirus. We found that both intracellular and extracellular recombinant protein production significantly increased when expression of caspase-1 is inhibited in BmN cells. These data indicate that blocking the apoptotic pathway is a promising way to enhance recombinant protein production in BEVSs.

A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

In-silico determination of Pichia pastoris signal peptides for extracellular recombinant protein production

In-silico identified novel secretory signal peptides (SPs) are required in vivo to achieve efficient transfer or to prevent other cellular proteins from interfering with the process in extracellular recombinant protein (r-protein) production. 56 endogenous and exogenous secretory SPs, have been used or having the potential to be used in Pichia pastoris for r-protein secretion, were analyzed in-silico using the softwares namely SignalP4.1, Phobius, WolfPsort0.2, ProP1.0, and NetNGlyc1.0. Among the predicted 41 endogenous secretory SPs, five of them have been used in P. pastoris, and regarded as positive controls; whereas, 36 of them have not been used. Amongst, the predicted cleavage site for each of the 32 endogenous secretory SPs was found to be same by the three programs. The secretory SPs having the highest D-scores, the score quantifying the signal peptide-ness of a given sequence segment, were: MKILSALLLLFTLAFA (D=0.932), MRPVLSLLLLLASSVLA (D=0.932), MFKSLCMLIGSCLLSSVLA (D=0.918). As D-scores of these SPs are higher than that of Saccharomyces cerevisiae α-mating factor signal peptide which has been widely used for r-protein production, they can be considered as the promising candidates. Among the predicted 15 exogenous SPs, 11 have been used in P. pastoris: therefore, they were evaluated as positive controls. The three programs predicted a unique cleavage site for each of the 10 exogenous SPs; and D-scores of these SPs were within D=0.805–0.900; whereas, four exogenous secretory SPs, which have not been used in P. pastoris, have D-scores within D=0.494–0.702.

Extracellular recombinant protein production under continuous culture conditions with Escherichia coli using an alternative plasmid selection mechanism

The secretion of recombinant proteins into the extracellular space by Escherichia coli presents advantages like easier purification and protection from proteolytic degradation. The controlled co-expression of a bacteriocin release protein aids in moving periplasmic proteins through the outer membrane. Since such systems have rarely been applied in continuous culture it seemed to be attractive to study the interplay between growth-phase regulated promoters controlling release protein genes and the productivity of a chemostat process. To avoid the use of antibiotics and render this process more sustainable, alternative plasmid selection mechanisms were required. In the current study, the strain E. coli JM109 harboring plasmid p582 was shown to stably express and secrete recombinant β-glucanase in continuous culture using a minimal medium. The segregational instability of the plasmid in the absence of antibiotic selection pressure was demonstrated. The leuB gene, crucial in the leucine biosynthetic pathway, was cloned onto plasmid p582 and the new construct transformed into an E. coli Keio (ΔleuB) knockout strain. The ability of the construct to complement the leucine auxotrophy was initially tested in shake-flasks and batch cultivation. Later, this strain was successfully grown for more than 200h in a chemostat and was found to be able to express the recombinant protein. Significantly, it showed a stable maintenance of the recombinant plasmid in the absence of any antibiotics. The plasmid stability in a continuously cultivated E. coli fermentation, in the absence of antibiotics, with extracellular secretion of recombinant protein provides an interesting model for further improvements.

Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli.

BackgroundChromobacterium violaceum is a free-living β-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the β-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum.ResultsThe open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa, as estimated by denaturing polyacrylamide gel electrophoresis. N-terminal sequencing confirmed the proper removal of the native signal peptide during the secretion of the recombinant product. The enzyme was able to hydrolyze colloidal chitin and the synthetic substrates p-nitrophenyl-β-D-N,N’-diacetylchitobiose and p-nitrophenyl-β-D-N,N’,N”-triacetylchitotriose. The optimum pH for its activity was 5.0, and the enzyme retained ~32% of its activity when heated to 60°C for 30 min.ConclusionsA C. violaceum chitinase was expressed in E. coli and purified by affinity chromatography on a chitin matrix. The secretion of the recombinant protein into the culture medium was directed by its native signal peptide. The mature enzyme was able to hydrolyze colloidal chitin and synthetic substrates. This newly identified signal peptide is a promising secretion factor that should be further investigated in future studies, aiming to demonstrate its usefulness as an alternative tool for the extracellular production of recombinant proteins in E. coli.

Effects of temperature shifts and oscillations on recombinant protein production expressed in Escherichia coli

Escherichia coli is widely used host for the intracellular expression of many proteins. However, in some cases also secretion of protein from periplasm was observed. Improvement of both intracellular and extracellular production of recombinant protein in E. coli is an attractive goal in order to reduce production cost and increase process efficiency and economics. Since heat shock proteins in E. coli were reported to be helpful for protein refolding and hindering aggregation, in this work different types of single and periodic heat shocks were tested on lab scale to enhance intracellular and extracellular protein production. A single heat shock prior to induction and different oscillatory temperature variations during the induction phase were executed. The results showed that these variations influence protein production negatively. In other words, 45 and 50 % reduction in extracellular protein production were observed for the single heat shock and oscillated temperature between 35 and 40 °C, respectively. However, the oscillatory temperature approach introduced in this study is recommended as a tool to quantitatively analyze the effects of inhomogeneous temperature on cell physiology and productivity in large-scale bioreactors.

Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.

Extracellular accumulation of recombinant protein by Escherichia coli in a defined medium

Extracellular accumulation of recombinant proteins in the culture medium of Escherichia coli is desirable but difficult to obtain. The inner or cytoplasmic membrane and the outer membrane of E. coli are two barriers for releasing recombinant proteins expressed in the cytoplasm into the culture medium. Even if recombinant proteins have been exported into the periplasm, a space between the outer membrane and the inner membrane, the outer membrane remains the last barrier for their extracellular release. However, when E. coli was cultured in a particular defined medium, recombinant proteins exported into the periplasm could diffuse into the culture medium automatically. If a nonionic detergent, Triton X-100, was added in the medium, recombinant proteins expressed in the cytoplasm could also be released into the culture medium. It was then that extracellular accumulation of recombinant proteins could be obtained by exporting them into the periplasm or releasing them from the cytoplasm with Triton X-100 addition. The tactics described herein provided simple and valuable methods for achieving extracellular production of recombinant proteins in E. coli.