Marina I. Lebedeva

Biotechnology Institute

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Sergey V. Mashko

Ajinomoto (Russia)

Roy A. Mariuzza

University of Maryland, College Park

ВП

В. П. Вейко

A N Bach Institute of Biochemistry

Alla Lapidus

Independent University of Moscow

Barry A. Fields

University of Southern Queensland

E. Sally Ward

University of Southampton

Andrea S. Llera

Fundación Instituto Leloir

Lara S. Izotova

Rutgers, The State University of New Jersey

Hongmin Li

China Tobacco

S M Podkovyrov

Virginia Tech

Cooperative Institutions

Genetika

Biotechnology Institute

University of Idaho

The University of Texas Southwestern Medical Center

University of Buenos Aires

National Institute of Standards and Technology

University of Minnesota

University of Minnesota Medical Center

Russian Academy of Sciences

Lomonosov Moscow State University

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Use of TGATG vector for expression of a cloned cDNA copy of human interleukin-1β gene in Escherichia coli cells

Sergey V. Mashko Sergei V. Kotenko В. П. Вейко Marina I. Lebedeva Alla Lapidus

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A Mutational Analysis of the Binding of Staphylococcal Enterotoxins B and C3 to the T Cell Receptor β Chain and Major Histocompatibility Complex Class II

The Journal of Experimental Medicine (1998)

Lukas Leder Andrea S. Llera Pascal M. Lavoie Marina I. Lebedeva Hongmin Li

The three-dimensional structure of the complex between a T cell receptor (TCR) β chain (mouse Vβ8.2Jβ2.1Cβ1) and the superantigen (SAG) staphylococcal enterotoxin C3 (SEC3) has been recently determined to 3.5 Å resolution. To evaluate the actual contribution of individual SAG residues to stabilizing the β–SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 and staphylococcal enterotoxin B (SEB) mutants to soluble recombinant TCR β chain and to the human MHC class II molecule HLA-DR1. Affinities were determined by sedimentation equilibrium and/or surface plasmon detection, while mitogenic potency was assessed using T cells from rearrangement-deficient TCR transgenic mice. We show that there is a clear and simple relationship between the affinity of SAGs for the TCR and their biological activity: the tighter the binding of a particular mutant of SEC3 or SEB to the TCR β chain, the greater its ability to stimulate T cells. We also find that there is an interplay between TCR–SAG and SAG–MHC interactions in determining mitogenic potency, such that a small increase in the affinity of a SAG for MHC can overcome a large decrease in the SAG's affinity for the TCR. Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the β–SEC3 complex (“hot spot” residues) are strictly conserved among enterotoxins reactive with mouse Vβ8.2, thereby providing a basis for understanding why SAGs having other residues at these positions show different Vβ-binding specificities.

Superantigen

10.1084/jem.187.6.823

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Citations (145)

[Expression of the chloramphenicol acetyltransferase gene is under control of various promoters of E. coli and phage lambda].

PubMed (1986)

Sergey V. Mashko S M Podkovyrov M E Trukhan Rena Gorovits Marina I. Lebedeva

Plasmids have been constructed with the structural region of the cat gene being under the control of the lactose (lacUV5), tryptophane (trpOP), operons of Escherichia coli, the hybrid trp-lac (tac) promoter and early bacteriophage lambda promoters (PL, PR and PLIT). The expression of chloramphenicolacetyltransferase gene in Escherichia coli cells harbouring such recombinant plasmids and pBR325 as well has been examined by determining the chloramphenicol resistance and studying the enzyme activity of Cm-acetylase. A high level of enzyme synthesis is connected with transcription from PL, PR and tac promoters.

Chloramphenicol acetyltransferase

Transcription

Acetyltransferases

Terminator (solar)

Lambda phage

Structural gene

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Citations (1)

Structure of the Vδ domain of a human γδ T-cell antigen receptor

Nature (1998)

Hongmin Li Marina I. Lebedeva Andrea S. Llera Barry A. Fields Michael B. Brenner

MHC restriction

10.1038/35172

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Citations (130)

Cloning, expression, and crystallization of the Vδ domain of a human γδ T‐cell receptor

Protein Science (1996)

Marina I. Lebedeva Barry A. Fields Hergen Spits Govindaswamy Panchamoorthy Michael B. Brenner

Abstract T‐lymphocytes recognize a wide variety of antigens through highly diverse cell‐surface glycoproteins known as T‐cell receptors (TCRs). These disulfide‐linked heterodimers are composed of α and β or γ and δ polypeptide chains consisting of variable (V) and constant (C) domains non‐covalently associated with at least four invariant chains to form the TCR‐CD3 complex. It is well established that αβ TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three‐dimensional structure of αβ TCRs has recently become available through X‐ray crystallography. In contrast, the antigen specificity of γδ TCRs is much less well understood and their three‐dimensional structure is unknown. We have cloned the δ chain of a human TCR specific for the MHC class I HLA‐A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli . Following affinity purification using a nickel chelate adsorbent, the recombinant Vδ domain was crystallized in a form suitable for X‐ray diffraction analysis. The crystals are orthorhombic, space group P 2 1 2 1 2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 Å, and diffract to beyond 2.3 Å resolution. The ability of a Vδ domain produced in bacteria to form well‐ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of γδ TCRs.

10.1002/pro.5560051226

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Citations (4)

[Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells. I. Construction of vectors for the cloning of transcription regulatory elements].

PubMed (1985)

Sergey V. Mashko Marina I. Lebedeva S M Podkovyrov Mikhail Kashlev M E Trukhan

New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments.

Chloramphenicol acetyltransferase

Multiple cloning site

Transcription

Cloning (programming)

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Crystal structure of a CDR3 mutant of a T cell antigen receptor Va domain

Acta Crystallographica Section A Foundations of Crystallography (1996)

H. Li Marina I. Lebedeva E. Sally Ward Roy A. Mariuzza

investigations.The structural information contributing to the elucidation of HBP's multifunctionality and the role of the heavy glycosylation.

10.1107/s0108767396090514

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Expression of the hAng gene in Escherichia coli; isolation and characterization of human recombinant Ser-(-1) angiogenin.

PubMed (1990)

Alla Lapidus A. V. Mochul'skii S M Podkovyrov Marina I. Lebedeva A A Antipin

Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.

Angiogenin

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Citations (2)

Crystal Structure of the V _α Domain of a T Cell Antigen Receptor

Science (1995)

Barry A. Fields Bertram T. Ober Emilio L. Malchiodi Marina I. Lebedeva Bradford C. Braden

The crystal structure of the V α domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin topology set different from those previously described. A switch in a polypeptide strand from one β sheet to the other enables a pair of V α homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable loops face in one direction. On the basis of the observed mode of V α association, a model of an (αβ) 2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (αβ) 2 tetramer with the third hypervariable loop of V α over the amino-terminal portion of the antigenic peptide and the corresponding loop of V β over its carboxyl-terminal residues. TCR dimerization that is mediated by the α chain may contribute to the coupling of antigen recognition to signal transduction during T cell activation.

Tetramer

Hypervariable region

BETA (programming language)

Immunoglobulin superfamily

10.1126/science.270.5243.1821

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Citations (179)

T7 RNA polymerase-derived expression of the human interleukin-8 cDNA in Escherichia coli. Purification and initial characterization of recombinant hIL-8

Биотехнология (1993)

Ivan B. Lomakin Marina I. Lebedeva В. Г. Конусова E.V. Velichko V.F. Lebedev

T7 RNA polymerase

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