Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae.

Recently the existence of 'heavy chain' immunoglobulins in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to haptens. In addition, it was not a priori predictable whether the binding domains (VHH) of these antibodies could be produced and secreted by the lower eukaryotic micro-organism Saccharomyces cerevisiae. In the present study these questions are addressed. Heavy chain immunoglobulins directed against two hapten molecules, the azo-dyes RR6 and RR120 as well as the (proteinaceous) human pregnancy hormone, have been raised in Lama glama. We were able to select specific VHH fragments for all three antigens by direct screening of Escherichia coli or yeast libraries, even without prior enrichment via bio-panning. This is the first example of the isolation of llama anti-hapten VHH domains. Surprisingly, the affinities of the llama VHHs for the RR6 hapten obtained in this way are in the low nM range. Furthermore, some of the antigen specific VHHs were secreted by S. cerevisiae at levels over 100 mg l-1 in shake flask cultures. These two findings extend the possible application areas for the llama VHH fragments significantly.

[1]  F. Klis,et al.  Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins , 1997, Applied and environmental microbiology.

[2]  Andrew J. Martin,et al.  Antibody-antigen interactions: contact analysis and binding site topography. , 1996, Journal of molecular biology.

[3]  L. Wyns,et al.  Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies , 1997, FEBS letters.

[4]  R. Lerner,et al.  Chemical Selection for Catalysis in Combinatorial Antibody Libraries , 1997, Science.

[5]  Lode Wyns,et al.  Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies , 1998, The EMBO journal.

[6]  J. Verbakel,et al.  Expression and secretion of antifreeze peptides in the yeast Saccharomyces cerevisiae , 1995, Yeast.

[7]  Eric T. Boder,et al.  Yeast surface display for screening combinatorial polypeptide libraries , 1997, Nature Biotechnology.

[8]  C. Verrips,et al.  Recent advances in the large-scale production of antibody fragments using lower eukaryotic microorganisms. , 1998, Research in immunology.

[9]  J. Cronin,et al.  The resonant mirror: a novel optical biosensor for direct sensing of biomolecular interactions Part I: Principle of operation and associated instrumentation , 1992 .

[10]  R. Raines,et al.  Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments , 1998, Nature Biotechnology.

[11]  F. Klis,et al.  Immobilizing proteins on the surface of yeast cells. , 1996, Trends in biotechnology.

[12]  E. van Tuinen,et al.  Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae. , 1993, Gene.

[13]  H R Hoogenboom,et al.  Designing and optimizing library selection strategies for generating high-affinity antibodies. , 1997, Trends in biotechnology.

[14]  H R Hoogenboom,et al.  Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. , 1991, Nucleic acids research.

[15]  L. Wyns,et al.  Comparison of llama VH sequences from conventional and heavy chain antibodies. , 1997, Molecular immunology.

[16]  G. Winter,et al.  Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Dominique Bourgeois,et al.  The crystal structure of a llama heavy chain variable domain , 1996, Nature Structural Biology.

[18]  A. Lesk,et al.  Canonical structures for the hypervariable regions of immunoglobulins. , 1987, Journal of molecular biology.

[19]  J. W. Bos,et al.  ScFv Antibody Fragments Produced in Saccharomyces cerevisiae Accumulate in the Endoplasmic Reticulum and the Vacuole , 1994 .

[20]  R. Schmitz,et al.  Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in the Yeast Pichia pastoris , 1995, Bio/Technology.

[21]  T. Scanlan,et al.  The structural and functional basis of antibody catalysis. , 1997, Annual review of biophysics and biomolecular structure.

[22]  E. Sasso,et al.  Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup. , 1991, Journal of immunology.

[23]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[24]  J. Duffus,et al.  Yeast : a practical approach , 1988 .

[25]  S. Muyldermans,et al.  Naturally occurring antibodies devoid of light chains , 1993, Nature.

[26]  M. Murtaugh,et al.  High-level secretion of two antibody single chain Fv fragments by Pichia pastoris. , 1997, Journal of immunological methods.

[27]  W. Harris,et al.  Separation and concentration of bacteria with immobilized antibody fragments. , 1995, The Journal of applied bacteriology.

[28]  Desmond G. Higgins,et al.  Fast and sensitive multiple sequence alignments on a microcomputer , 1989, Comput. Appl. Biosci..

[29]  E. Padlan,et al.  Anatomy of the antibody molecule. , 1994, Molecular immunology.

[30]  C T Verrips,et al.  Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. , 1999, Biochimica et biophysica acta.

[31]  P. Dunnill,et al.  Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli. , 1997, Biotechnology and bioengineering.

[32]  A. Plückthun,et al.  High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions , 1996, Applied Microbiology and Biotechnology.

[33]  Brad Snedecor,et al.  High Level Escherichia coli Expression and Production of a Bivalent Humanized Antibody Fragment , 1992, Bio/Technology.

[34]  E. Claassen,et al.  Adjuvant properties of stable water-in-oil emulsions: evaluation of the experience with Specol. , 1992, Research in immunology.