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Biomedical subjects

J Ghrayeb

Publications and source records attributed to J Ghrayeb.

At least 37 records · Page 2Linked to original sources
PubMed1992

Stable expression of cloned human antibody genes in murine myeloma cells.

Human monoclonal antibodies (MAbs) offer potential advantages over murine MAbs for therapy because they are not likely to elicit immune responses and are expected to interact more efficiently with the human immune system to activate therapeutically useful functions. Traditional methods for obtaining human MAbs (i.e., immortalization of B cells by cell fusion or transformation) can result in low and unstable antibody secretion. Recently, methods have been devised for direct cloning of human variable region genes via polymerase chain reaction and phage combinatorial libraries. Both types of human MAb production can benefit from expression systems that support the stable, high-level antibody secretion required for therapeutic use. Using an existing human-derived hybridoma that secretes a human IgM antibody as a convenient source of antibody genes, we have demonstrated that cloned human antibody genes can be efficiently expressed in murine myeloma cells and that cell lines with properties suitable for large-scale economical production can be obtained. We were unable to detect any differences between the antibodies produced by the original hybridoma and the engineered cell line. In addition, we were able to express an IgG form of the antibody, showing that expression of a recombinant human antibody need not be limited to the original antibody class.

Animals↗
PubMed1992

High-level expression and characterization of a mouse-human chimeric CD4 antibody with therapeutic potential.

The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 micrograms/ml chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells.

Animals↗
PubMed1990

Pharmacokinetics of a mouse/human chimeric monoclonal antibody (C-17-1A) in metastatic adenocarcinoma patients.

The pharmacokinetic characteristics of a mouse/human chimeric monoclonal antibody (C-17-1A) were determined in 10 patients with metastatic adenocarcinoma following the administration of either 10-mg or 40-mg infusions as a single or multiple dose. The administration of single 10-mg (n = 5) and 40-mg (n = 5) doses infused over 1 hr resulted in mean apparent steady-state distribution volumes of 4.13 +/- 0.97 and 5.16 +/- 1.92 liters, respectively, indicating that C-17-1A appears to distribute throughout the vascular compartment and into limited extracellular fluid volume. The disposition of C-17-1A was adequately characterized using a two-compartment open model with mean distribution half-lives of 15.8 and 18.5 hr and mean elimination half-lives of 90.0 and 97.6 hr for the 10- and 40-mg groups, respectively. A linear relationship was observed between AUC and dose (micrograms/kg). The clearance of C-17-1A was correlated linearly with total Ig, IgG, and tumor size. Multiple administration of either 10-mg (n = 3) or 40-mg (n = 3) doses of C-17-1A infused over 1 hr every 14 days for a total of three doses resulted in superimposable mean serum concentration versus time data and consistent mean pharmacokinetic characteristics. These data indicate that C-17-1A exhibits linear, nonsaturable distribution and elimination characteristics in man over the dose range studied (i.e., 130 to 880 micrograms/kg). The multiple-dose pharmacokinetics of C-17-1A were predictable, indicating a lack of an antibody response to C-17-1A over a period of 42 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1990

Localization of B-cell stimulatory activity of HIV-1 to the carboxyl terminus of gp41.

Patients with AIDS are known to have B-cell hyperactivity. We have previously demonstrated that an extract of HIV-1 could induce differentiation of peripheral blood B lymphocytes of healthy volunteers into immunoglobulin-secreting cells. In an attempt to delineate the B-cell stimulatory subregion in HIV-1, we have investigated the influences of native glycoproteins and recombinant proteins of the envelope. The complete envelope glycoprotein, gp160 and a recombinant protein in the carboxyl terminal region of gp41 termed PE-8 were effective in inducing terminal differentiation of normal peripheral blood B lymphocytes and did so in a T-lymphocyte-dependent manner. The activity was not present in the native exterior envelope glycoprotein, gp120 and several other recombinant proteins, viz PE-2 an PE-3, which are in the amino terminal region of gp120 or in env-9, a protein in the junctional region of gp120 and gp41. Polyclonal and monoclonal antibodies directed to diverse regions of the envelope abrogated the influence of gp160. The PE-8-induced B-cell differentiation was abrogated by goat anti-gp160 antibody but not by goat anti-gp120 antibody or monoclonal antibody to the amino terminal of gp41. These studies suggest that a putative polyclonal B-cell stimulatory epitope of HIV-1 is located in the carboxyl end of the envelope glycoprotein.

B-Lymphocytes↗
PubMed1989

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources.

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.

Adult↗
PubMed1989

Biological characterization of a chimeric mouse-human IgM antibody directed against the 17-1A antigen.

A chimeric antibody was constructed in which the murine H- and L-chain variable regions of mAb 17-1A, raised against human colorectal cancer cells, were joined with the human constant mu and kappa regions. Transfection of these constructs into the murine myeloma Sp2/0 resulted in the expression and secretion of a pentameric Ig, designated chimeric 17-1A IgM. The chimeric 17-1A IgM was subsequently compared to a previously described chimeric 17-1A IgG1 for biological activities. Both chimeric mAbs were equally effective (weight basis) in competing against the binding of murine 125I-17-1A to cultures of HT-29 colon carcinoma cells. The calculated association constants for the chimeric 17-1A IgM and IgG1 were 1.63 x 10(8) l/mol and 3.41 x 10(7) l/mol, respectively. Unlike chimeric 17-1A IgG1, the chimeric 17-1A IgM was able to render colon carcinoma target cells susceptible to lysis by both xenogeneic (rabbit) and human complement. The extent of complement-mediated lysis dependent upon chimeric 17-1A IgM was correlated to 17-1A antigen expression on target cells. HT-29 colon carcinoma cells treated with chimeric 17-1A IgM did not directly result in antibody-dependent cellular cytotoxicity by human peripheral blood monocytes. However, chimeric 17-1A IgM greatly enhanced the deposition of C3 on complement-treated HT-29 cells, and concomitant incubation with monocytes resulted in heightened lysis of the tumor cells. The feasibility of enhancing host defense against gastrointestinal malignancies by the administration of this chimeric 17-1A IgM may have certain clinical advantages.

Animals↗
PubMed1989

Post-translational modifications of human interleukin-6.

We have previously reported that interleukin (IL)-6 secreted by human fibroblasts induced with either IL-1 or tumor necrosis factor (TNF) consists of at least six differentially modified phosphoglycoproteins of molecular mass 23-30 kDa: a triplet in the mass range from 23 to 25 kDa and another triplet in the range from 28 to 30 kDa. We now report that a combination of metabolic labeling, glycosidase digestion, and lectin chromatography experiments demonstrates that the 23- to 25-kDa species are O-glycosylated and that the 28- to 30-kDa species are both O- and N-glycosylated. Pulse-chase experiments reveal that newly synthesized IL-6 polypeptides rapidly enter two separate protein modification pathways: one leads to O-glycosylation and the other to both N- and O-glycosylation; polypeptides in both pathways are further modified (phosphorylation) prior to secretion. Although both pathways appear to be equally utilized in IL-1- or TNF-induced fibroblasts, the relative proportion of polypeptides proceeding through one or the other pathway can be experimentally modified. In the presence of tunicamycin, IL-6 is secreted exclusively in the O-glycosylated form, whereas in the presence of cycloheximide the pathway leading to both N- and O-glycosylation is dominant. The inclusion of monensin (1 microM) does not inhibit IL-6 secretion from fibroblasts even though it inhibits glycosylation. Combined immunoprecipitation, immunoblotting, and immunoaffinity chromatography experiments reveal additional IL-6 species with mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions corresponding to molecular masses 17-19 kDa and 45 kDa, suggesting that this cytokine undergoes further alterations. These observations highlight an aspect of IL-6 biosynthesis that appears to represent an excellent model system for studying the mechanisms regulating post-translational protein modifications in human cells and also suggest a basis for reconciling conflicting descriptions of IL-6 structure.

Cell Line↗
PubMed1989

Mouse/human chimeric monoclonal antibody in man: kinetics and immune response.

A mouse/human chimeric monoclonal antibody (mAb) composed of the variable regions of murine 17-1A mAb and the constant regions of human IgG-1K immunoglobulin was administered to 10 patients with metastatic colon cancer. Four patients received single infusion of 10 mg (n = 2) or 40 mg (n = 2). Six patients received three infusion of 10 mg (n = 3) or 40 mg (n = 3) at 2-week intervals. The pharmacokinetics were similar at both dose levels and at the second and third infusions. The plasma disappearance curves were best fit by a two-compartment model having a mean alpha T1/2 of 17.5 hr (range 13-23 hr) and a mean beta T1/2 of 100.5 hr (range 65-139 hr). One patient who received three 40-mg doses of chimeric IgG-1K 17-1A mAb (day 0, 14, and 28) was the only patient to exhibit a detectable but modest antibody reactivity to chimera on days 63 and 84. The antibody reactivity was inhibited by murine 17-1A mAb, indicating that the antibody response was directed to the murine variable region of the chimera. The patients had no toxic or allergic reactions. This chimeric form of 17-1A mAb has an approximate 6-fold longer circulation time and appears to be substantially less immunogenic than its murine counterpart. These characteristics may provide an advantage in the clinical application of such chimeric molecules in therapeutic trials in humans.

Adenocarcinoma↗
PubMed1989

T-cell response towards HIV in infected individuals with and without zidovudine therapy, and in HIV-exposed sexual partners.

HIV-specific T-cell response in HIV-infected individuals at different stages of the disease and during zidovudine therapy was studied using HIV and HIV-envelope derived native and recombinant proteins as antigens. Neither antibody-negative at-risk individuals nor HIV-infected individuals responded to HIV or its envelope-derived proteins, even though they responded to a recall antigen, purified protein derivative of tuberculin (PPD). However, five out of 14 antibody- and antigen-negative sexual partners of known HIV-positive men did respond to HIV, native gp 120 and recombinant envelope and core proteins. Some AIDS-related complex (ARC) and AIDS patients treated with zidovudine also showed a low T-cell response which diminished along with clinical deterioration. A synthetic peptide representing one of the major T-cell epitopes in HIV envelope, frequently recognized by immunized and infected primates, gave only marginal stimulation in man. Our findings suggest that HIV infection in man results in a T-helper cell anergy directed against viral proteins. The response observed in the antibody- and antigen-negative sexual partners and in some of the zidovudine-treated patients implies that at least some epitopes on HIV envelope are immunogenic in man.

Acquired Immunodeficiency Syndrome↗
PubMed1988

The expression in Escherichia coli of recombinant human platelet factor 4, a protein with immunoregulatory activity.

In order to establish more firmly the immunoregulatory effect of platelet factor 4 (PF4) and develop a means to provide material for possible clinical use, the nucleotide sequence for PF4 was synthesized utilizing a ligation strategy of six duplexes ranging from 27 to 43 base pairs in length. The individual oligodeoxynucleotides were synthesized on an automated system. The resultant gene segment (226 base pairs), which incorporated convenient HindIII and BamHI overhangs at the 5' and 3' ends, respectively, was cloned into the pIN-III-ompA-2-expression vector in Escherichia coli, affording a fusion protein of Mr = 8900 with 7 additional amino acids at the amino terminus and 4 at the carboxyl terminus and with aspartic acid rather than asparagine in position 47. The recombinant PF4 (rPF4) was purified by heparin-agarose affinity chromatography and reverse-phase high performance liquid chromatography. It reacted with a monoclonal mouse anti-human PF4 antibody on a Western blot and in an enzyme-linked immunosorbent assay. The rPF4 protein exhibited an immunoregulatory effect like that of human PF4 in its ability to reverse concanavalin A-induced immunosuppression in BALB/c mice.

Amino Acid Sequence↗
PubMed1988

Synthesis and secretion of multiple forms of beta 2-interferon/B-cell differentiation factor 2/hepatocyte-stimulating factor by human fibroblasts and monocytes.

The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2). When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity. We have prepared a rabbit polyclonal antiserum to the E. coli-derived human IFN-beta 2 fusion protein. This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations. The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes. "Uninduced" human FS-4 fibroblasts as well as those induced with interleukin-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions. The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30). Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by [3H]glucosamine (gp23-gp25). The inclusion of cycloheximide during the [35S]methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet. Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells. Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.

Animals↗
PubMed1988

Regulation of the acute phase and immune responses in viral disease. Enhanced expression of the beta 2-interferon/hepatocyte-stimulating factor/interleukin 6 gene in virus-infected human fibroblasts.

We have defined the expression of the mRNA for, and secretion of, IFN-beta 2/hepatocyte-stimulating factor/IL-6 (IFN-beta 2/IL-6) in human diploid fibroblasts (FS-4 strain) infected with different RNA- and DNA-containing viruses. RNA blot-hybridization analyses carried out 6-8 h after the beginning of infection showed that the RNA-containing Sendai virus (paramyxoviridae) enhanced IFN-beta 2/IL-6 mRNA levels 10-fold, followed, in decreasing order, by encephalomyocarditis (EMC, picornaviridae), vesicular stomatitis (VSV, rhabdoviridae), Newcastle disease virus (NDV, paramyxoviridae), and influenza A (Flu, myxoviridae) viruses. The DNA-containing pseudorabies virus (PR, herpesviridae) enhanced IFN-beta 2/IL-6 mRNA levels sixfold, while the effect of adenovirus type 5 (Ad5, adenoviridae) was considerably less and comparable with that of NDV or Flu. A rabbit antiserum raised against E. coli-derived human IFN-beta 2/IL-6 was used in immunoprecipitation experiments to monitor the secretion of 35S-methionine-pulse-labeled IFN-beta 2/IL-6 proteins by fibroblasts up to 7 h after the beginning of infection. Enhanced levels of secretion of IFN-beta 2/IL-6 (2-14-fold) were observed in every instance evaluated (Sendai, EMC, VSV, Flu, PR, Ad5 viruses). A biological consequence of enhanced secretion of IFN-beta 2/IL-6 was the ability of media from infected FS-4 cell cultures to enhance by 8-15-fold the synthesis and secretion of a typical acute phase plasma protein (alpha 1-antichymotrypsin) by human hepatoma Hep3B2 cells. These observations make it likely that IFN-beta 2/IL-6 mediates, in part, the host response to acute virus infections.

Acute-Phase Reaction↗
PubMed1988

Phosphorylation of secreted forms of human beta 2-interferon/hepatocyte stimulating factor/interleukin-6.

"Beta 2-Interferon/hepatocyte stimulating factor/interleukin-6" (IFN-beta 2) has emerged as a major mediator of the plasma protein response to tissue injury (the acute phase response) in addition to its numerous effects on cells of the immune system. Human fibroblasts and monocytes induced with tumor necrosis factor, interleukin-1, bacterial lipopolysaccharide (endotoxin) or virus infection secrete multiple forms of differentially glycosylated IFN-beta 2 polypeptides: at least a doublet of molecular mass approximately 25 kD and a triplet of mass approximately 30 kD. We report that immunoprecipitation analyses of medium from [32P]orthophosphate- labeled cultures of induced fibroblasts carried out using a rabbit polyclonal antibody to recombinant E. coli-derived human IFN-beta 2 reveal that the secreted gp23-25 and gp28-30 forms of IFN-beta 2 are phosphorylated. IFN-beta 2 gp23-25 secreted by induced monocytes is phosphorylated whereas the monocytic gp28-30 is poorly labeled with [32P]orthophosphate suggesting tissue-specific differences in IFN-beta 2 phosphorylation. Phosphoamino acid analyses indicate that all of the detected phosphate is in phosphoserine residues. Furthermore, IFN-beta 2 can be completely dephosphorylated by alkaline phosphatase (E.C. No. 3.1.3.1); thus all of the phosphate label is in readily accessible sites. These observations suggest the possibility that differential phosphorylation of IFN-beta 2 forms may be a mechanism to modulate its functions in a tissue-specific manner.

Alkaline Phosphatase↗
PubMed1988

Biological activity of human-mouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal antibodies with antitumor specificity.

Chimeric antibodies were constructed in which the murine variable region of anti-colorectal cancer monoclonal antibody CO17-1A was joined with human gamma 1, gamma 2, gamma 3, and gamma 4 constant regions. Human-mouse chimeric proteins were compared with the parental murine IgG2a antibody CO17-1A for their ability to participate in tumor-cell destruction by human and murine effector cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. All of the chimeric antibodies showed different degrees of ADCC with human lymphocytes, monocytes, and granulocytes and with murine macrophages. Monocytes and macrophages were able to utilize the chimeric IgG1 and, to a lesser degree, IgG4 and IgG3 antibodies to lyse tumor-cell targets in ADCC assays. The chimeric IgG1 and IgG4 antibodies were nearly as effective as the parental CO17-1A antibody in inhibiting tumor growth in nude mice. These data indicate that chimeric IgG1 antibody is superior in its antitumor activity.

Animals↗
PubMed1988

Nonrandom distribution of antibodies to the TRS protein of human immunodeficiency virus in infected people with different clinical status.

A transregulatory gene, trs, of human immunodeficiency virus I (HIV-1) was expressed in bacteria as a 26-kD fusion protein. Survey of over 100 individuals infected with HIV revealed a nonrandom distribution of seropositivity against trs: a few of the asymptomatic carriers and AIDS patients (less than 5%) had sera that reacted with the 26-kD protein. In contrast, 29% of the ARC patients' sera reacted positively. This result is different from those of serological reactivities of the other accessory gene products of HIV-1 (tat, sor, 3' orf, and R) which did not differentiate among stages of clinical progression. Since ARC is a prodrome for full-blown AIDS, these results suggest that trs may be useful as a prognostic marker for AIDS development.

AIDS-Related Complex↗
PubMed1988

Human lymphocyte and monocyte lysis of tumor cells mediated by a mouse/human IgG1 chimeric monoclonal antibody.

The mouse monoclonal antibody 17-1A (gamma 2a, kappa) recognizes a tumor-associated antigen expressed on human gastrointestinal malignancies and has been used in Phase I and II clinical trials. Chimeric genetic constructs have been produced using 17-1A variable region genes (VL and VH) and the constant region genes for human kappa light chains and gamma 1 heavy chains (C kappa and gamma 1). The chimeric gene constructs were transfected into mouse myeloma cells for antibody production. The secreted mouse/human chimeric antibody contains the antigen-binding domain of 17-1A and the human (gamma 1, kappa) constant regions. Native mouse 17-1A and the chimeric antibody (chIgG1) were analyzed for binding to two human colon cancer cell lines and for the mediation of cancer cell line antibody-dependent cellular cytotoxicity by normal human peripheral blood lymphocytes and monocytes in 4 h 51Cr-release assays. The 17-1A and chIgG1 gave similar results in these in vitro biologic assays. This study demonstrates the feasibility of using mouse/human chimeric antibodies in human therapeutic applications.

Animals↗
PubMed1987

Wild type and mutant signal peptides of Escherichia coli outer membrane lipoprotein interact with equal efficiency with mammalian signal recognition particle.

The signal peptide of the outer membrane lipoprotein (OMLP) of Escherichia coli was shown to be capable of promoting protein translocation across mammalian microsomal membranes in vitro. We assayed translocation of a fusion protein containing the OMLP signal peptide and nine amino acids of OMLP fused in frame to beta-lactamase. The efficiency with which the mammalian translocation machinery recognizes and accepts the OMLP signal peptide as substrate is indistinguishable from that of mammalian secretory proteins. Upon translocation mammalian signal peptidase processes the pre-OMLP-beta-lactamase protein at different sites than are utilized in vivo by E. coli OMLP signal peptidase (signal peptidase II) but that can be predicted as mammalian signal peptidase cleavage sites. Mutants in the OMLP signal peptide were tested for their ability to promote translocation of the fusion protein in this assay system. It has been shown previously that mutants in the positively charged amino acids at the amino terminus of the signal peptide severely delay the translocation of OMLP in vivo in E. coli. However, these mutants had no detectable effect either on signal recognition by mammalian signal recognition particle or on the efficiency of translocation itself.

Amino Acid Sequence↗