Obtaining recombinant forms of the outer membrane protein F of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and assessment of their immunogenic properties

Two recombinant forms of the outer membrane protein F (OprF) of Pseudomonas aeruginosa were obtained, the full-length protein OprF and the C-terminal part of the OprF protein (aa 192–342). As a result of double immunizations, these recombinant proteins provided mice with resistance to experimental intraperitoneal challenge with P. aeruginosa. The best protective effects were observed at a dose of 25 μg for OprF and 50 μg for the truncated OprF variant (indices of efficiency were 3.3 and 2.8, respectively). Rabbit antisera immune to the recombinant proteins were also able to protect mice from the experimental infection with P. aeruginosa. Indices of efficiency were 6.4 for OprF and 6.0 for the OprF C-terminal part; these values were approximately two times as high as the effect of sera from intact test animals (3.2).     

Preparation of Pseudomonas aeruginosa recombinant outer-membrane protein F and the study of its antigenic properties

In Escherichia coli M15, the gene of P. aeruginosa recombinant outer-membrane protein F (OprF) was cloned. OprF, chromatographically purified on Ni-agarose and containing an additional sequence of 6 histidines on the N-end, was obtained. The purified OprF specifically reacted with rabbit serum, hyperimmune to P. aeruginosa, and in the mice injected with this protein specific IgG antibodies were synthesized. The optimum concentrations of P. aeruginosa OprF were selected for further tests of its protective properties from infection induced by P. aeruginosa.     

Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.     

Involvement of the C-terminal part of Pseudomonas fluorescens OprF in the modulation of its pore-forming properties

The major outer-membrane protein, OprF, from the psychrotrophic bacterium Pseudomonas fluorescens undergoes a reduction of its conductance value (from 250 pS to 80 pS) when the growth temperature is shifted from 28 degrees C to 8 degrees C. The involvement of changes in tertiary or quaternary structure in this behaviour, was implied by enzymatic digestion experiments in which OprFs purified from 8 degrees C and 28 degrees C cultures showed different accessibility to pronase. Resistant proteolytic fragments of 19 kDa, obtained from both OprF preparations, were identified as the N-terminal half of the native protein. These 19 kDa fragments induced ion channels in planar lipid bilayers with similar conductance values of 65-75 pS in 1 M NaCl, in contrast to the native proteins. Thus, the C-terminal part of the protein is required for the growth temperature-dependent modulation of OprF channel-forming properties. LPS was not detected on the proteolytic fragments while it was found in similar amounts on the native OprFs. These results suggest the LPS/porin association occurs through the C-terminal part of the porin. Radiolabelling experiments showed different phosphorylation levels of LPS for 8 degrees C and 28 degrees C cultures. Thus, in response to growth temperature, the structural modification of the LPS could be associated to the modulation of OprF pore size.     

Overexpression and purification of PreS region of hepatitis B virus antigenic surface protein adr subtype in Escherichia coli

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80 degrees.     

羧端部分缺失的HCV核心蛋白的融合表达,免疫原性及其应用

构建了丙型肝炎病毒核心蛋白的全长及Ｎ端和Ｎ端与谷胱甘肽巯基转移酶（ＧＳＴ）的融合表达克隆,比较了在不同大肠杆菌中的表达。表达蛋白为水溶性,经ＥＬＩＳＡ和蛋白质印迹分析,ＧＳＴＣ１９１的表达和稳定性都较差,ＧＳＴＣ６９和ＧＳＴＣ４０具有良好的稳定性,用ＧＳＴ亲和柱一步纯化,纯度可达９０％,免疫小鼠可产生高滴度的抗体。应用表达的ＧＳＴＣ６９和ＧＳＴＣ４０抗原,检测人血清中的ＨＣＶ核心蛋白抗体,初步结果     

SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定

为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据。     

Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.     

Isolation and characterization of biologically active murine interleukin-6 produced in Escherichia coli.

Interleukin-6 (IL-6) is a multi-functional cytokine produced and secreted by several different cell types, including those of the immune system. A cDNA coding for the mature murine IL-6 (mIL-6), which extends from amino acid (aa) 25 through 211, was cloned into a prokaryotic vector and then expressed in Escherichia coli. The recombinant mIL-6 (remIL-6) was isolated from bacterial inclusion bodies by solubilization in 4 M guanidine hydrochloride followed by gel-filtration chromatography. The protein was refolded to an active conformation by dialysis against 25 mM Na. acetate pH 5.5. A final step of purification and concentration on a cation exchange resin yielded pure and biologically active remIL-6. The purified preparation had the expected aa composition, as confirmed by aa analysis and pI of 7.0-7.1. The biological activity of the recombinant protein was measured in two systems; a proliferation assay employing 7TD1 cells, and a fibrinogen biosynthesis assay employing primary rat hepatocytes. Both assay systems demonstrated that the remIL-6 was active in the range of 10(8) units/mg, which is similar to that estimated for native cytokine. Antibodies raised in rabbits against remIL-6 neutralized the biological activity of both recombinant and native IL-6.     

Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.     

Biotechnological production of acetylated thymosin β4

Thymosin β4 (43 aa) is a highly conserved acidic peptide, which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin β4 is undergoing clinical trials as a drug for treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin β4 is produced by a solid-phase chemical synthesis. Biotechnological synthesis of this peptide is difficult, because the N-terminal amino acid residue of thymosin β4 playing an essential role in the actin interaction is acetylated. In this study, we proposed a method for production of a thymosin β4 recombinant precursor and its directed chemical acetylation. Deacetylthymosin β4 was synthesized as a part of a hybrid protein containing thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for purification of deacetylthymosin β4: (i) biosynthesis of a soluble hybrid protein (HP) in Escherichia coli, (ii) isolation of HP by ion exchange chromatography, (iii) cleavage of HP with TEV protease, and (iv) purification of deacetylthymosin β4 by ultrafiltration. N-Terminal acetylation of the serine residue of deacetylthymosin β4 was performed with acetic anhydride under acidic conditions (pH 3.0). The reaction yield was 55%. Thymosin β4 was finally purified by reverse-phase HPLC. The proposed method of isolation of recombinant thymosin β4 can be scaled-up and provide a highly purified preparation in a yield of 20 mg per 1 L of culture suitable for use in medical practice.     

化学合成虎纹捕鸟蛛毒素-I基因的克隆和表达

本文报道了全化学合成虎纹捕鸟蛛毒素－Ⅰ基因在大肠杆菌中的表达,表达产物为Ｎ－端是谷胱甘肽硫转移酶的融合蛋白．经ＧＳＨ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化,凝血酶酶解融合蛋白,得到重组ＨＷＴＸ－Ⅰ（ｒＨＷＴＸ－Ⅰ）．质谱和氨基酸顺序分析均表明ｒＨＷＴＸ－Ⅰ系正确表达产物．还原复性的ｒＨＷＴＸ－Ⅰ表现出与天然ＨＷＴＸ－Ⅰ生物学活性的一致性．     

Biotechnological production of acetylated thymosin beta4

Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.     

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH(2)-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (k(cat)/K(m)) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H(2)O(2), which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.     

Immunochemical properties of West Nile virus protein prM and protein M C-end

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag

The efficiencies of different procedures for purification of the capsid protein (CA) of Mason-Pfizer monkey virus are compared. Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for affinity chromatography purification were prepared. CA was expressed in Escherichia coli (i) as a wild-type protein, (ii) C-terminally extended with a six-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (CA 6aa6His). Electron microscopy was used for comparison of the resulting proteins, as CA is a structural protein with no enzymatic activity. We have found that these C-terminal fusions dramatically influenced the properties and morphology of structures formed by CA protein in E. coli. The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His proteins formed organized structures. CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form. Both six-histidine-tagged proteins were purified using affinity chromatography under either native (CA 6His) or denaturing (CA 6aa6His) conditions. CA protein was purified under denaturing conditions using gel-filtration chromatography followed by refolding. All proteins were obtained at a purity >98%. Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form organized structures within E. coli. We show here that the widely used histidine anchor may significantly alter the properties of the protein of interest.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Expression, secretion, and glycosylation of the 45- and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

The gene encoding the 45/47 kDa glycoprotein (Rv1860) of Mycobacterium tuberculosis was expressed in Streptomyces lividans under its own promoter and under the thiostrepton-inducible Streptomyces promoter PtipA. The recombinant protein was released into the culture medium and, like the native protein, migrated as a double band at 45 and 47 kDa in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels. However, in contrast to the native protein, only the 47-kDa recombinant protein could be labeled with concanavalin A (ConA). Carbohydrate digestion with jack bean alpha-D-mannosidase resulted in a reduction in the molecular mass of the recombinant protein upper band and completely eliminated ConA binding. Two-dimensional gel electrophoresis revealed only one isoelectric point for the recombinant protein. Comparative fingerprinting analysis of the individually purified upper and lower recombinant protein bands, treated under the same conditions with specific proteases, resulted in similar peptide patterns, and the peptides had the same N-terminal sequence, suggesting that migration of the recombinant protein as two bands in SDS-PAGE gels could be due to differences in glycosylation. Mass spectrometry analysis of the recombinant protein indicated that as in native protein, both the N-terminal and C-terminal domains of the recombinant protein are glycosylated. Furthermore, it was determined that antibodies of human tuberculosis patients reacted mainly against the carbohydrate residues of the glycoprotein. Altogether, these observations show that expression of genes for mycobacterial antigens in S. lividans is very useful for elucidation of the functional role and molecular mechanisms of glycosylation in bacteria.     

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1.

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.