Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Inhibition of the activating signals in NK92 cells by recombinant GST-sHLA-G1α chain

The soluble HLA-G1 (sHLA-G1) isoform was found to be secreted by trophoblast cells at the materno-fetal interface,which suggests that it may act as an immunomodulator during pregnancy. In this paper, we reported that GST-sHLA-G1α chain could bind to its receptor ILT-2 on NK92 cells and then the latter recruited Src homology 2 domaincontaining tyrosine phosphatase-1 (SHP-1), which consequently dephosphorylated some important protein tyrosine kinases and blocked the activation of downstream molecules such as MEK and ERK so that the cytotoxicity of natural killer (NK) cells was inhibited. These results indicated that GST-sHLA-G1α chain might be exploited in new immunotherapy strategies aiming at inducing immunotolerance during allograft, xenograft and autoimmune situations. In addition,we found that modification of O-linked β-N-acetylglucosamine (O-GlcNAc) was involved in NK cells‘ activating and inhibitory signals. This may provide a novel molecular target for inducing immunotolerance but needs further study.     

Pseudohypoparathyroidism type Ia: two new heterozygous frameshift mutations in exons 5 and 10 of the Gsα gene

Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disease characterized by resistance to PTH and other hormones that act via cAMP. Patients have deficient activity of Gs, the subunit of the G protein, which couples hormone receptors to stimulation of adenylate cyclase. We describe two new mutations discovered in two sporadic patients with PHP-Ia. Using genomic DNA, we have amplified exons 2–13 of the Gs gene (GNAS1) by PCR, and sequenced the resulting products. Both patients had Albright's hereditary osteodystrophy, resistance to multiple hormones, and deficient Gs activity. In the first patient, a deletion of a C in exon 5 at codon 115 was found. In the second patient, an insertion of a C in exon 10 at codon 267 was detected. Both these heterozygous mutations cause frameshift, and predict decreased production of Gs. This report adds two new Gs mutations to the known ten mutations recently described.     

Proline excretion in Escherichia coli: A comparison of an argD + strain and a proline-excreting argD − derivative

In an attempt to deduce the physiological basis of proline excretion in argD ^– strains of Escherichia coli K12, several properties of an argD ⁺ (nonexcreting) and an argD ^– (excreting) derivative were compared. No difference was found in the transport or in the utilization of either proline or its immediate precursor, ¹-pyrroline-5-carboxylate (PCA). Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase. The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD ^– mutant.This work was supported by grants from the National Institutes of Allergy and Infectious Diseases (Public Health Service No. AI-10862) and The University of Connecticut Research Foundation (to C. M. B.). J. J. R. was supported by an NDEA Predoctoral Fellowship.     

Efficient E. coli expression systems for the production of recombinant β-mannanases and other bacterial extracellular enzymes

Two Escherichia coli expression systems based on T7 RNA polymerase promoter (pET system) and tac promoter (pFLAG system) have been used for the production and secretion of recombinant β-mannanases from Bacillus sp. Both E. coli OmpA signal peptide and native Bacillus signal peptide could be used efficiently for the secretion of recombinant enzymes into periplasmic space and culture media. The genes could be induced for over-expression with 0.1-1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) when the OD 600 of the culture broth reached 0.6-1.5. The recombinant enzymes could be harvested from whole cell lysate, perimplasmic extract, or culture broth after induction for 4-20 hours. Since the enzyme is C-terminally tagged with hexahistidine, the recombinant enzymes could be conveniently purified to apparent homogeneity by one-step immobilized-metal affinity chromatography (IMAC) using Ni-NTA resins. The characteristics of purified recombinant β-mannanases from B. licheniformis and B. subtilis, which share 78% amino acid identity, are slightly different. These systems should be applicable for the production of various recombinant bacterial extracellular enzymes.     

Variable synthesis and expression of E β and A e (E β ) Ia polypeptide chains in mice of differentH-2 haplotypes

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice of thed, k, p, andr haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, E and A_e (E ). Cells from mice of theb ands haplotypes fail to synthesize E chains but do synthesize A_e chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of thef andq haplotypes fail to synthesize either the A_e or E polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact E :A_e complex are consistent with the theory that both the A_e and E polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics ofI-region-controlled functions are discussed.     

A new strategy for secretory expression and mixed fermentation of recombinant human collagen α1 (III) chain in Pichia pastoris

Recombinant human full-length mature collagen α1 (III) chain (rhCOL3A1) was secreted by Pichia pastoris GS115, using the Saccharmyces cerevisiae á-mating factor prepro signal, and the theoretical molecular weight of rhCOL3A1 was 95.344 kDa. The gene cloned from human placenta, was designed and cloned into expression vector pPIC9K under the control of a strong inducible promoter AOX1.The expression stage of rhCOL3A1 was sensitive to different carbon ratios through mixed fermentation. LCMS/ MS analysis and western blotting demonstrated that the recombinant human full-length mature collagen a1 (III) gene was successfully expressed in P. pastoris GS115 during the methanol induction stage. Furthermore, an effective strategy of mixed fermentation was established to express rhCOL3A1 in shake flash. Compared to single carbon induction, when induced with mixed carbon at the ration of 0.8 (glycerol/methanol), the time corresponding to the highest yield of rhCOL3A1 (1.27 g/L) was drastically reduced by 50%. The same conclusion was observed from RT-qPCR. Consequently, a new strategy which was more time-saving and effective was provided for the large-scale producing the full-length mature rhCOL3A1.     

Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system

The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult‐to‐express proteins. For this reason, we evaluated the more recently established novel CAP‐T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP‐T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. Biotechnol. Bioeng. 2012;109: 2250–2261. © 2012 Wiley Periodicals, Inc.     

Isolation of a Δ6-desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaena sp.strain PCC 7120

The enzyme ⁶-desaturase is responsible for the conversion of linoleic acid (18:2) to -linolenic acid (18:3). A cyanobacterial gene encoding ⁶-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking ⁶-desaturase. Expression of the Synechocystis ⁶-desaturase gene in Anabaena resulted in the accumulation of -linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis ⁶-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a ¹²-desaturase gene linked to the ⁶-desaturase gene. Expression of Synechocystis ⁶-and ¹²-desaturase in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and -linolenic acid.     

L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression

Abstract

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch^®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch^® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.     

Matrix metalloproteinase-dependent microsomal prostaglandin E synthase-1 expression in macrophages: role of TNF-α and the EP4 prostanoid receptor

Matrix metalloproteinase (MMP)-9 contributes to the pathogenesis of chronic inflammatory diseases and cancer. Thus, identifying targetable components of signaling pathways that regulate MMP-9 expression may have broad therapeutic implications. Our previous studies revealed a nexus between metalloproteinases and prostanoids whereby MMP-1 and MMP-3, commonly found in inflammatory and neoplastic foci, stimulate macrophage MMP-9 expression via the release of TNF-α and subsequent induction of cyclooxygenase-2 and PGE(2) engagement of EP4 receptor. In the current study, we determined whether MMP-induced cyclooxygenase-2 expression was coupled to the expression of prostaglandin E synthase family members. We found that MMP-1- and MMP-3-dependent release of TNF-α induced rapid and transient expression of early growth response protein 1 in macrophages followed by sustained elevation in microsomal prostaglandin synthase 1 (mPGES-1) expression. Metalloproteinase-induced PGE(2) levels and MMP-9 expression were markedly attenuated in macrophages in which mPGES-1 was silenced, thereby identifying mPGES-1 as a therapeutic target in the regulation of MMP-9 expression. Finally, the induction of mPGES-1 was regulated, in part, through a positive feedback loop dependent on PGE(2) binding to EP4. Thus, in addition to inhibiting macrophage MMP-9 expression, EP4 antagonists emerge as potential therapy to reduce mPGES-1 expression and PGE(2) levels in inflammatory and neoplastic settings.     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Cloning and expression of sucrose phosphorylase gene from Bifidobacterium longum in E. coli and characterization of the recombinant enzyme

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.