Functional expression of Candida antarctica lipase B in Eschericha coli

Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants k(M), v(max) and k(cat) were determined using the substrates p-NPB and p-NPL. Almost identical k(cat)/k(M) values (0.423-0.466 min(-1) microM(-1) for p-NPB and 0.068-0.071 min(-1) microM(-1) for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.     

白地霉ch-3脂肪酶Ⅰ基因在大肠杆菌中的表达

构建了白地霉脂肪酶Ⅰ的基因工程菌,为进一步进行蛋白质工程改造和脂肪酶应用奠定了基础。从新疆昌吉市油脂化工厂含油冻土中分离得到1株低温脂肪酶产生菌-白地霉ch-3。该菌发酵上清液中的脂肪酶最适作用温度为35℃,在0℃仍可保持66%的相对酶活力。应用PCR技术从白地霉ch-3基因组DNA中克隆得到脂肪酶Ⅰ基因lip1,将该基因与原核表达质粒载体pET-22b（＋）连接,构建重组质粒pETl-ip1,转化E.coliBL21（DE3）,酶切鉴定,筛选得到重组菌。十二烷基磺酸钠-聚丙希酰胺（SDS-PAGE）显示重组脂肪酶Ⅰ的相对分子质量约为5.8×10^4,酶活为2.73 U/mL,表明lip1基因的表达产物具有正常的生物学活性。白地霉ch-3脂肪酶Ⅰ基因lip1能够在大肠杆菌中有效地表达。     

Soluble expression of Candida antarctica lipase B in Escherichia coli by fusion with Skp chaperone

Candida antarctica lipase B (CalB) is an industrially versatile enzyme, especially for biodiesel production and organic synthesis. Recombinant expression using the E. coli system has advantages, such as lower costs, easier handling, and higher number of clones that can be screened daily compared to expression using higher organism. But the expression of CalB in E. coli is not feasible because insoluble aggregates are formed and proteolytic degradation is known to occur during expression. In this study, fusion proteins were designed to express soluble CalB in E. coli. The periplasmic chaperone of E. coli, Skp was fused with CalB and this fusion protein showed a high solubility (yielding 82.5 ??g/mL). The fusion protein system can be applied to the rapid expression and evaluation of CalB variants for functional improvement.     

Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

Preparation of recombinant glycoprotease of Pasteurella haemolytica A1 utilizing the Escherichia coliα-hemolysin secretion system

Abstract Three murine monoclonal antibodies were prepared against the recombinant glycoprotease of Pasteurella haemolytica A1 expressed in Escherichia coli . These monoclonal antibodies were able to recognize the authentic glycoprotease from P. haemolytica A1 culture supernatant. A recombinant plasmid which contained most of the glycoprotease gene of P. haemolytica A1 fused with the secretion signal sequence from hlyA of the E. coli α-hemolysin determinant was constructed. This recombinant plasmid expressed a fusion protein (Gcp-F) which was secreted into the culture supernantant by E. coli cells when the α-hemolysin secretion functions HlyB and HlyD are supplied in trans. Gcp-F could be readily recovered from the supernatant free from other cellular materials and is suitable for use in vaccine trials and challenge experiments in animals.     

pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production

The production of recombinant protein in Escherichia coli is often hampered by low expression levels and low solubility. A variety of methodologies have been developed including protein production at low temperature, and fusion protein expression using soluble protein tags. Here, we present the novel cold-shock vector pCold-GST for high-level expression of soluble proteins in E. coli. This vector is a modified pCold I cold-shock vector that includes the glutathione S-transferase (GST) tag. The pCold-GST expression system developed was applied to 10 proteins that could not be expressed using conventional E. coli expression methodologies, and nine of these proteins were successfully obtained in the soluble fraction. The expression and purification of two unstable protein fragments were also demonstrated by employing a C-terminal hexa-histidine tag for purification purposes. The purified proteins were amenable to NMR analyses. These data suggest that the pCold-GST expression system can be utilized to improve the expression and purification of various proteins.     

Cell-free synthesis and multifold screening of Candida antarctica lipase B (CalB) variants after combinatorial mutagenesis of hot spots

We have developed a strategy for rapid and combinatorial optimization of the hot spot residues of enzymes. After combinatorial randomization of target locations in the Candida antarctica lipase B (CalB) gene, the individual variant genes isolated in the E.coli cells were expressed in the cell-free protein synthesis system to analyze different parameters of the resulting CalB variants. The enzymatic assays for the hydrolysis of para-nitrophenyl-ester (pNP-ester) and triglyceride, synthesis of wax ester, and thermal stability of the variant enzymes were carried out simultaneously in 96-well microtiter plates. From the 1,000 variant genes tested in each assay, we were able to identify a series of the variant enzymes having markedly improved hydrolytic, synthetic activity, or thermal stability. The improved traits of the cell-free selected CalB variants were well reproduced when the corresponding genes were expressed in Pichia pastoris. Therefore, we expect that the proposed strategy of cell-free expression screening can serve as a viable option for rapid and precise tuning of enzyme molecules, not only for analytical purposes but also for industrial applications through large scale production using microbial cells transformed with variant genes selected from the cell-free expression screening.     

Enhanced soluble protein expression using two new fusion tags

Production of soluble recombinant proteins is vital for structure-function analysis and therapeutic applications. Unfortunately, when expressed in a heterologous host, such as Escherichia coli, most proteins are expressed as insoluble aggregates. Two new fusion partners have been identified to address these solubility problems. One of the tags was derived from a bacteriophage T7 protein kinase and the other one from a small E. coli chaperone, Skp. We have expressed a panel of insoluble human proteins including Hif1alpha, IL13, and folliculin as fusion proteins using these tags. Most of these fusion proteins were able to be expressed in a soluble form and could be purified by virtue of a Strep-tag II installed at the amino-terminal end of the fusion partners. In addition, we show that some of these proteins remained soluble after removal of the fusion tags by a site-specific protease. The results with these tags compare favorably to results with the most commonly used solubility tags described in the literature. Therefore, these two new fusion tags have the potential to express soluble proteins when fused with many recalcitrant proteins.     

Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function

High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Engineering Escherichia coli BL21(DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography

Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The "NiCo" strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.     

Expression of honeybee prepromelittin as a fusion protein in Escherichia coli.

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.     

杆状病毒表达系统的改进及IL-6在昆虫细胞内的表达和纯化

使用同源重组方法,在昆虫细胞内将多角体启动子驱动的EGFP表达盒插入杆状病毒穿梭载体Bacmid的p74位相,经5轮空斑纯化获得重组穿梭载体Bacmid-egfp。然后将Bacmid-egfp转化含转座助手质粒的E.coliDH10B,获得受体菌E.coliDH10Bac-egfp,由于Bacmid-egfp保留了完整的转座结构和α互补功能,因此该菌株和原始E.coliDH10Bac一样能有效的利用各种pFastBac系列的载体进行转座并构建出能指示病毒繁殖和目的基因表达的重组病毒。使用红色荧光蛋白DsRed对系统进行了验证,结果表明重组病毒Bac-egfp-DsRed感染的细胞中绿色荧光蛋白和红色荧光蛋白均得到了高效表达。进一步使用该系统在昆虫细胞中高效表达并纯化了IL-6蛋白,为研究和应用该细胞因子提供物质基础,同时也进一步证明所改造的杆状病毒表达系统的可靠性和实用性。     

Two methods for improved purification of full-length mammalian proteins that have poor expression and/or solubility using standard Escherichia coli procedures

Many mammalian proteins are multifunctional proteins with biological activities whose characterization often requires in vitro studies. However, these studies depend on generation of sufficient quantities of recombinant protein and many mammalian proteins cannot be easily expressed and purified as full-length products. One example is the Wilm's tumor gene product, WT1, which has proven difficult to express as a full-length purified recombinant protein using standard approaches. To facilitate expression of full-length WT1 we have developed approaches that optimized its expression and purification in Escherichia coli and mammalian cells. First, using a bicistronic vector system, we successfully expressed and purified WT1 containing a C-terminal tandem affinity tag in 293T cells. Second, using a specific strain of E. coli transformed with a modified GST vector, we successfully expressed and purified N-terminal GST tagged and C-terminal 2x FLAG tagged full-length human WT1. The benefits of these approaches include: (1) two-step affinity purification to allow high quality of protein purification, (2) large soluble tags that can be used for a first affinity purification step, but then conveniently removed with the highly site-specific TEV protease, and (3) the use of non-denaturing purification and elution conditions that are predicted to preserve native protein conformation and function.     

Recombinant expression of the Candida rugosa lip4 lipase in Escherichia coli

It is difficult to express recombinant Candida rugosa lipases (CRLs) in heterologous systems, since C. rugosa utilizes a nonuniversal serine codon CUG for leucine. In this study, recombinant LIP4 in which all 19 CUG codons had been converted to a universal serine codon was overexpressed in Escherichia coli BL21(DE3). The recombinant LIP4 was found mainly in the inclusion bodies and showed a low catalytic activity. To increase the amount of soluble form and activity of recombinant LIP4, the DNA was fused to the gene for thioredoxin (TrxFus-LIP4) and then expressed in E. coli strain AD494(DE3). This strategy promotes the formation of disulfide bonds in the cytosol and yields enzymatically active forms of LIP4. The purified recombinant TrxFus-LIP4 and LIP4 expressed in AD494(DE3) had the same catalytic profiles. In addition, recombinant LIP4 had higher esterase activities toward long-chain ester and lower lipase activities toward tributyrin, triolein, and olive oil. This system for the expression of fungal lipase in E. coli strain AD494(DE3) is reliable and may produce enzymatically active forms of recombinant lipase without an in vitro refolding procedure.     

A strategy for the expression of recombinant proteins traditionally hard to purify

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.     

抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。