High level expression of recombinant Mycobacterium tuberculosis culture filtrate protein CFP32 in Pichia pastoris.

Difficulty in obtaining large quantities of Mycobacterium tuberculosis (MTB) proteins remains a major obstacle in the development of subunit vaccines and diagnostic reagents for tuberculosis. A major reason is because Escherichia coli has not proven to be an optimal host for the expression of MTB genes. In this article, we used the yeast Pichia pastoris to express high levels of CFP32, a culture filtrate protein restricted to the MTB complex and a potential target antigen for serodiagnosis of tuberculosis in patients. Using shaker flasks, we generated a P. pastoris clone expressing CFP32 as a secreted protein fused to the myc- (His)6 tag, at a yield of 0.5 g of purified protein per liter of culture. Recombinant CFP32 (rCFP32) produced in P. pastoris has a molecular weight of 35 kDa, which is slightly higher than that of the native protein. We identified putative acylation and glycosylation sites in the CFP32 amino acid sequence that suggested posttranslational modifications may contribute to the size difference. The NH2-terminal peptide sequencing of rCFP32 showed that the signal peptide alpha factor is correctly excised. In addition, rCFP32 reacted with the sera of patients with tuberculosis. These data are the first to show that P. pastoris is a suitable host for high-yield production of good quality mycobacterium antigens, and especially culture filtrate proteins that have vaccine and diagnostic potential.

Recombinant integrin CD11b A-domain blocks polymorphonuclear cells recruitment and protects against skeletal muscle inflammatory injury in the rat.

The beta2 integrin CD11b/CD18 (CR3) is a major adhesion receptor of neutrophils, normally utilized to fend off infections. This receptor contributes, however, to multiple forms of non-infectious inflammatory injury when dysregulated as shown in gene knock-outs and through the use of blocking monoclonal antibodies. The major ligand recognition site of CR3 has been mapped to the A-domain in the CD11b subunit (CD11bA). The recombinant form of this domain exhibits a ligand binding profile similar to that of the holoreceptor. To assess the potential anti-inflammatory activity of CD11bA as a competitive antagonist of CR3 in vivo, we assessed its effects on a developed animal model of traumatic skeletal muscle injury in the rat. Recombinant soluble rat CD11bA-domain fused to glutathione-S-transferase (GST) was administered intravenously in a single dose at 1 mg/kg to nine groups of Wistar rats, five in each group, 30 min before inducing traumatic skeletal muscle injury. Control animals received either a function-blocking anti-CD11b/CD18 monoclonal antibody (1 mg/kg), non-functional mutant forms of the CD11bA (D140GS/AGA, T209/A, D242/A), recombinant GST or buffer alone. In control animals, the wounded muscle showed oedema, erythrocyte extravasation and myonecrosis both within and outside the immediate wounded area (5-10 mm zone) and influx of neutrophils was detected 30 min post-wound, followed by a second wave 3 hr later. Wild-type CD11bA- or anti-CD11b monoclonal antibody (mAb)-treated rats showed a comparable and significant decrease in the number of infiltrating PMN (78 + 4%, n = 70 and 86 +/- 2%, n = 50, respectively) and preservation of the muscular fibres outside the immediate zone of necrosis (75 + 4%, n = 70, 84 +/- 1%, n = 50, respectively), compared to controls. These data demonstrate that CD11bA can be an effective tissue-preserving agent in acute inflammatory muscular injury.

Non-radioactive detection of five common microsatellite markers for ATP7B gene in Wilson disease patients.

Haplotype analysis using microsatellite markers is a useful indicator of specific mutations and is often exploited as the first large-scale screening technique to carry out the molecular characterization of the disease gene in probands from a specific population. However, the methodologies available are still cumbersome and require the use of either radioactive compounds or specialized equipment suitable to follow fluorescent dyes. Both these techniques may not be available for newly developing clinical laboratories. We have set up a sensitive and easy-to-use protocol to characterize five closely spaced, highly polymorphic microsatellite polymorphisms (CA repeats) that span the Wilson disease (WD) region, i.e. D13S316, D13S133, D13S301, D13S314, D13S315. The technique described here for the analysis of the WD gene microsatellite system relies on the quick detection method of silver staining, avoiding the use of toxic or sophisticated equipment. This approach could be the method of choice to implement molecular genetic testing in clinical laboratories, even those not especially equipped for DNA analysis and in particular in newly developed molecular genetics centers in countries whose population has not yet been characterized for WD-causing ATP7B gene mutations.

Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules.

The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.

Two Novel Frame Shift, Recurrent and De Novo Mutations in the ITGB2 (CD18) Gene Causing Leukocyte Adhesion Deficiency in a Highly Inbred North African Population.

We have identified four different mutations causing leukocyte adhesion Deficiency (LAD) in the ITGB2 gene of patients from a highly inbred population. Two were novel single-bp deletions (1497delG and 1920delG) causing frame shift and the two others were the missense mutations G284S and R593C. In our study, the G284S was a recurrent mutation while the R593C occurred de novo. We have also characterized a novel Xba1 polymorphic site located at the 5' end of the ITGB2 locus. Family studies showed that the 1497delG mutation segregated with this marker and the intragenic AvaII polymorphic marker, suggesting the presence of a founder effect. The observation of a heterogeneous spectrum including de novo and recurrent mutations causing LAD in a highly inbred population is rather unexpected. In view of the literature published on the molecular genetics of LAD and considering the ethnic origin of the patients studied, our findings confirm the heterogeneity of the mutations causing LAD and point out potential mutational hot spots in the ITGB2 gene.

A model to study the effects of a viral inactivator (beta-propiolactone) on DNA ligation and gene expression in E. coli and Cos cells.

An experimental model to study the effects of viral inactivators on the biological properties of DNA was developed. Beta-propiolactone (betaPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that betaPL impairs these two major DNA functions are presented. The amounts of betaPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations.

Carbonic anhydrase II (CA II) deficiency in Maghrebian patients: evidence for founder effect and genomic recombination at the CA II locus.

A splice junction mutation at the exon 2-intron 2 boundary of the carbonic anhydrase II (CA II) gene was previously shown to be the unique mutation underlying the CA II deficiency syndrome in patients of Arab descent. Fourteen Tunisian (Maghrebian) families with a history of osteopetrosis, renal tubular acidosis, mental retardation, and CA II deficiency were studied to test the hypothesis that the mutation, found in all 24 patients, derived from a common ancestor originating in the Arabic Peninsula. A filiation study permitted us to trace these families back to a common Arabic tribe that settled in the Maghreb in the tenth century, indicating a common ethnic origin for these families. Segregation of the mutation with a TaqI biallelic restriction site polymorphism upstream of the CA II gene was studied by sequence-tagged site analysis in all the family members. These studies showed cosegregation of the Taq (-) allele with the mutation in 12 families out of 14. This observation supports a founder effect to explain the common CA II deficiency allele in this population. In the remaining two families, a genomic recombination or gene conversion occurred between the TaqI restriction marker and the mutation causing the disease. The relatively high recombination frequency suggests the presence of a hot spot for recombination or gene conversion at the CA II locus.

A unique mutation underlying carbonic anhydrase II deficiency syndrome in patients of Arab descent.

We have investigated, in the genomic DNA of ten Tunisian patients, the presence of a splice junction mutation at the 5' end of intron 2 in the carbonic anhydrase II gene (CAII) previously described in six CAII-deficient patients presumed to be of Arab origin. All our patients were homozygous for this mutation and were mentally retarded, a characteristic feature of the phenotype of patients with an Arabic background. This mutation is found exclusively in patients with an Arabic background and thus may be confined to this ethnic group.

Molecular cloning of a novel human hsp70 from a B cell line and its assignment to chromosome 5.

A 2391-bp cDNA encoding a novel human hsp70, named hsp70 RY, is described. It was cloned from a cDNA library constructed using mRNA derived from an established EBV-transformed B cell line from a patient with leukocyte adhesion molecule deficiency. hsp70 RY is 701 amino acids long, has the characteristic N-terminal ATP-binding domain and the C-terminal peptide binding domain, and contains four potential N-glycosylation sites. Northern blotting revealed a single mRNA species of 3.0 kb in total RNA prepared from the patient's EBV cell line. In situ hybridization localized the single copy hsp70 RY gene to the long arm of chromosome 5 at 5 q31.1-5 q31.2.

Expression of a soluble and functional form of the human beta 2 integrin CD11b/CD18.

Polymorphonuclear cells and monocytes (phagocytes) are a critical component of host defense against infections. However, these cells also play a significant role in host tissue damage in many noninfectious diseases, such as ischemia-reperfusion injury syndromes and rejection of transplanted organs. The leukocyte adhesion molecule family CD11/CD18 (beta 2 integrins) is critical to the function of polymorphonuclear cells and monocytes in inflammation and injury. Inherited deficiency of CD11/CD18 impairs phagocyte chemotaxis, adhesion and transmigration across endothelium, and clearance of invading microorganisms through phagocytosis and cell-mediated killing. Furthermore, murine monoclonal antibodies directed against the CD11b/CD18 (CR3) heterodimer have been shown to reduce, by 50%-80%, phagocyte-mediated ischemia-reperfusion injury in several organ systems, such as the myocardium, liver, and gastrointestinal tract and to inhibit development of insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice. Expression of CD11b/CD18 in a soluble and functional form might therefore be potentially useful as an anti-inflammatory agent. We have now expressed a recombinant soluble heterodimeric form of this human beta 2 integrin, normally expressed as two noncovalently associated membrane-bound subunits. The secreted receptor exhibited direct and specific binding to its ligand, iC3b, the major complement C3 opsonin, and inhibited binding of polymorphonuclear cells to recombinant interleukin 1-activated endothelium.

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

Point mutations impairing cell surface expression of the common beta subunit (CD18) in a patient with leukocyte adhesion molecule (Leu-CAM) deficiency.

The leukocyte adhesion molecules CD11a/CD18, CD11b/CD18, and CD11c/CD18 (Leu-CAM) are members of the integrin receptor family and mediate crucial adhesion-dependent functions in leukocytes. The molecular basis for their deficient cell surface expression was sought in a patient suffering from severe and recurrent bacterial infections. Previous studies revealed that impaired cell surface expression of Leu-CAM is secondary to heterogeneous structural defects in the common beta subunit (CD18). Cloning and sequencing of complementary DNA encoding for CD18 in this patient revealed two mutant alleles, each representing a point mutation in the coding region of CD18 and resulting in an amino acid substitution. Each mutant allele results in impaired CD18 expression on the cell surface membrane of transfected COS M6 cells. One substitution involves an arginine residue (Arg593----cysteine) that is conserved in the highly homologous fourth cysteine-rich repeats of other mammalian integrin subfamilies. The other substitution involves a lysine residue (Lys196----threonine) located within another highly conserved region in integrins. These data identify crucial residues and regions necessary for normal cell surface expression of CD18 and possibly other integrin beta subunits and define a molecular basis for impaired cell surface expression of CD18 in this patient.