High yield expression and purification of recombinant human apolipoprotein A-II in Escherichia coli

Recombinant expression systems have become powerful tools for understanding the structure and function of proteins, including the apolipoproteins that comprise human HDL. However, human apolipoprotein (apo)A-II has proven difficult to produce by recombinant techniques, likely contributing to our lack of knowledge about its structure, specific biological function, and role in cardiovascular disease. Here we present a novel Escherichia coli-based recombinant expression system that produces highly pure mature human apoA-II at substantial yields. A Mxe GyrA intein containing a chitin binding domain was fused at the C terminus of apoA-II. A 6× histidine-tag was also added at the fusion protein's C terminus. After rapid purification on a chitin column, intein auto-cleavage was induced under reducing conditions, releasing a peptide with only one extra N-terminal Met compared with the sequence of human mature apoA-II. A pass through a nickel chelating column removed any histidine-tagged residual fusion protein, leaving highly pure apoA-II. A variety of electrophoretic, mass spectrometric, and spectrophotometric analyses demonstrated that the recombinant form is comparable in structure to human plasma apoA-II. Similarly, recombinant apoA-II is comparable to the plasma form in its ability to bind and reorganize lipid and promote cholesterol efflux from macrophages via the ATP binding cassette transporter A1. This system is ideal for producing large quantities of recombinant wild-type or mutant apoA-II for structural or functional studies.     

Efficient expression and purification of recombinant glutaminase from Bacillus licheniformis (GlsA) in Escherichia coli

Glutaminase or L-glutamine aminohydrolase (EC 3.5.1.2) is an enzyme that catalyzes the formation of glutamic acid and ammonium ion from glutamine. This enzyme functions in cellular metabolism of every organism by supplying nitrogen required for the biosynthesis of a variety of metabolic intermediates, while glutamic acid plays a role in both sensory and nutritional properties of food. So far there have been only a few reports on cloning, expression and characterization of purified glutaminases. Microbial glutaminases are enzymes with emerging potential in both the food and the pharmaceutical industries. In this research a recombinant glutaminase from Bacillus licheniformis (GlsA) was expressed in Escherichia coli, under the control of a ptac promoter. The recombinant enzyme was tagged with decahistidine tag at its C-terminus and could be conveniently purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The enzyme could be induced for efficient expression with IPTG, yielding approximately 26,000 units from 1-l shake flask cultures. The enzyme was stable at 30°C and pH 7.5 for up to 6h, and could be used efficiently to increase glutamic acid content when protein hydrolysates from soy and anchovy were used as substrates. The study demonstrates an efficient expression system for the production and purification of bacterial glutaminase. In addition, its potential application for bioconversion of glutamine to flavor-enhancing glutamic acid has been demonstrated.     

Expression and purification of recombinant human alpha-defensins in Escherichia coli

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.     

Large scale expression and purification of recombinant RNA in Escherichia coli

Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.     

Expression and purification of recombinant human annexin V in Escherichia coli

Human annexin V cDNA was cloned into plasmid pET19b and fused to a ten consecutive histidine tag at N-terminal. When expressed in E. coli BL21(DE3) LysS, the recombinant His10-annexin V accumulated in soluble form in the cytoplasm. By two-step chromatography, i.e., metal chelate affinity chromatography and anion exchange chromatography, recombinant His10-annexin V was purified to homogeneity on silver-stained SDS-PAGE gel. Recombinant annexin V, 7.4 mg, was obtained from a 1 litre flask culture.     

Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II

L-Asparaginase (isozyme II) from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. Extracellular expression of recombinant asparaginase was obtained by fusing the gene coding for asparaginase to an efficient pelB leader sequence and an N-terminal 6x histidine tag cloned under the T7lac promoter. Media composition and the induction strategy had a major influence on the specificity and efficiency of secretion of recombinant asparaginase. Induction of the cells with 0.1 mM IPTG at late log phase of growth in TB media resulted in fourfold higher extracellular activity in comparison to growing the cells in LB media followed by induction during the mid log phase. Using an optimized expression strategy a yield of 20,950 UI/L of recombinant asparaginase was obtained from the extracellular medium. The recombinant protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography which gave an overall yield of 95 mg/L of purified protein, with a recovery of 86%. This is approximately 8-fold higher to the previously reported data in literature. The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.     

Efficient expression and purification of human aglycosylated Fcγ receptors in Escherichia coli

Effector Fc gamma receptors (FcγRs) are expressed on the surface of a variety of cells of hematopoietic lineage and serve as a bridge between adaptive and innate immune responses. The interaction between immune complexes, formed by IgG class antibodies that are crosslinked with antigen, and FcγRs triggers signaling cascades that result in numerous cellular responses including the activation or donwregulation of cytotoxic responses, cytokine release, and antibody synthesis. Here, the extracellular domains of the human type I transmembrane FcγRs were expressed in Escherichia coli and their interactions to subclass IgGs (IgG1, IgG2, IgG3, and IgG4) antibodies were analyzed. Expression using fully synthetic E. coli codon optimized FcγR genes and optimization of sequences for N‐terminal translation initiation region through mRNA secondary structure prediction enabled us to achieve high yield of purified, bacterially expressed receptors, including FcγRI and FcγRIIIa which have not been successfully expressed in bacteria until now. The aglycosylated FcγRs showed similar IgG subclass binding selectivity compared to the respective glycosylated FcγRs expressed in mammalian cells. Biotechnol. Bioeng. 2010;107: 21–30. © 2010 Wiley Periodicals, Inc.     

Efficient expression system of human recombinant laminin-5

Laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, is an essential component of various epithelial basement membranes, and it strongly promotes cellular adhesion and motility in vitro. In this study, we established an efficient expression system of human recombinant laminin-5 (rLN5), in which full-length cDNAs encoding the human laminin alpha3, beta3, and gamma2 chains were introduced into the human embryonic kidney cell line HEK293. rLN5 was purified from the conditioned medium of the HEK293 transfectant (LN5-HEK) by immuno-affinity chromatography in a yield of 1 mg protein/liter, about 10 times higher than that of a natural LN5 from human gastric cancer cells. rLN5 was indistinguishable from the natural LN5 in its protein composition and biological activity. In addition, analysis of HEK293 transfectants expressing two exogenous LN5 subunits showed that the alpha3/gamma2 chains and the beta3/gamma2 chains, but not the alpha3/beta3 chains, were secreted as heterodimers, suggesting an important role of the gamma2 chain in the association of the three LN5 subunits. The expression system of rLN5 can be used as an important tool to understand the biological functions of this laminin and may be applicable to future regenerative medicine.     

Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase.     

重组人尿激酶原在CHO细胞中的高效表达及其纯化

用鸡β- globin的MAR序列和人看家基因延伸因子1α(hEF-1α)的调控序列以及旱獭RNA稳定与输出序列,构建了重组人尿激酶原(recombinant human pro-urokinase,rhPro - UK)的高效表达载体,在CHO细胞中获得了rhPro - UK的高效稳定表达,rhPro - UK的表达水平达到1299 IU(以百万细胞1d的表达量计).采用阳离子交换层析、疏水层析和凝胶排阻层析的三步工艺纯化表达rhPro - UK的CHO细胞培养上清液,rhPro - UK的纯度达到98％、回收率为60％ ～70％.     

Expression and purification of recombinant human interleukin-18 protein using a yeast expression system

Interleukin-18 (IL-18) has been reported to exert significant immunoregulatory effects on inhibiting tumor growth through stimulating natural killer (NK) cell cytotoxicity and promoting production of several cytokines, including interferon-gamma (IFN-gamma) and granulocyte/macrophage colony-stimulating factor (GM-CSF). Therefore, IL-18 might serve as a potential therapeutic target for cancer treatment. However, the resource of this protein limits its availability for the clinical practice. The purpose of this study was to express and purify recombinant human (h) IL-18 protein using a yeast expression system. We reported here that hIL-18 gene was cloned into pPICZaC vector for expressing a recombinant hIL-18 protein using a yeast expression system. The recombinant hIL-18 protein was purified using centrifugal filter devices, hydrophobic chromatography, and anion exchange chromatography. The yield and purity of the recombinant hIL-18 reached 45.1% and 97.6%, respectively. This recombinant hIL-18 was shown to induce IFN-gamma production by human peripheral blood mononuclear cells (PBMCs) and enhance NK cell cytotoxicity synergistically with IL-2. Furthermore, these recombinant hIL-18-induced effects were the same as those by standard hIL-18. Therefore, the yeast expression system used in this study provides a useful method to produce large-scale of hIL-18 for the clinical application.     

Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli

Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.     

A new method for purification of recombinant human alpha-synuclein in Escherichia coli

alpha-Synuclein (AS), a major component of Lewy body in Parkinson's disease patients, exists as a natively unfolded protein in physiological buffer. We recently found that the overexpressed AS in Escherichia coli bearing the cloned AS cDNA with no signal sequence was actually located inside the periplasm, but not in the cytoplasm as generally recognized. Therefore, a new protocol for preparing recombinant AS has been developed with only two steps: (1) osmotic shock for release of AS-containing periplasm fraction and (2) ion-exchange chromatography for further purification of AS. By using plasmids and E. coli strains commonly used the new protocol is much more convenient, faster, and cheaper compared to the current methods established since 1994. About 80 mg AS with 95% purity can be regularly prepared from a 1L culture in 3 days.     

Overexpression and purification of recombinant human interferon alpha2b in Escherichia coli

Overexpression of rhIFN-alpha2b was obtained by synthesizing a codon optimized gene for IFN-alpha2b and expressing it in the form of inclusion bodies (IBs) in Escherichia coli. The recombinant plasmid pRSET-IFNalpha, which had the IFN-alpha2b gene under the T7 promoter, was coexpressed with plasmid pGP1-2, which carried the gene for T7 RNA polymerase under the heat inducible lambdaP(L) promoter. This two plasmid expression system was optimized with respect to heat shock time, media, and time of induction in shake flask cultures. This was then scaled up into a bioreactor to get a maximum volumetric product yield of 5.2g/L at a final OD(600) of 67. At this point, the IBs represented approximately 40% of the total cellular protein. This high specific product yields eased the further downstream processing steps and improved product recoveries. The IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of approximately 3g/L, which is maximum reported in the literature. The bioassay of the refolded protein gave a specific activity of approximately 3 x 10(9)IU/mg protein.     

Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli

The lymphokine human interleukin 4 (IL-4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL-4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL-4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel-filtration column. The refolded human IL-4 was characterized by N-terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli-derived human IL-4 has biological activity on T and B cells and binds to the human IL-4 receptor, comparable to mammalian expressed human IL-4, indicating that the protein is folded correctly.     

Expression and purification of recombinant neurotensin in Escherichia coli

An expression system has been designed for the rapid and economic expression of recombinant neurotensin for biophysical studies. A synthetic gene for neurotensin (Glu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)-Arg(9)-Pro(1 0)-Tyr(11)-Ile(12)-Leu(13)) was cloned into the pGEX-5X-2 vector to allow expression of neurotensin as a glutathione S-transferase (GST) fusion protein. The inclusion of a methionine residue between the glutathione S-transferase and the neurotensin has facilitated the rapid cleavage of the neurotensin from its carrier protein. Purification of recombinant neurotensin was performed by reverse-phase HPLC. This method produced a relatively high yield of peptide and offers the potential for economic partial or uniform labeling of small peptides (<15 amino acids) with isotopes for NMR or other biophysical techniques.