Isolation of Llama Antibody Fragments for Prevention of Dandruff by Phage Display in Shampoo

ABSTRACT As part of research exploring the feasibility of using antibody fragments to inhibit the growth of organisms implicated in dandruff, we isolated antibody fragments that bind to a cell surface protein of Malassezia furfur in the presence of shampoo. We found that phage display of llama single-domain antibody fragments (VHHs) can be extended to very harsh conditions, such as the presence of shampoo containing nonionic and anionic surfactants. We selected several VHHs that bind to the cell wall protein Malf1 of M. furfur, a fungus implicated in causing dandruff. In addition to high stability in the presence of shampoo, these VHHs are also stable under other denaturing conditions, such as high urea concentrations. Many of the stable VHHs were found to contain arginine at position 44. Replacement of the native amino acid at position 44 with arginine in the most stable VHH that lacked this arginine resulted in a dramatic further increase in the stability. The combination of the unique properties of VHHs together with applied phage display and protein engineering is a powerful method for obtaining highly stable VHHs that can be used in a wide range of applications.

[1]  G. Donnarumma,et al.  Possible role of Malassezia furfur in psoriasis: modulation of TGF‐β1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis‐affected patients , 2004, Journal of cutaneous pathology.

[2]  C T Verrips,et al.  Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography. , 2003, Biochimica et biophysica acta.

[3]  Frank Grosveld,et al.  Intracellularly Expressed Single-Domain Antibody against p15 Matrix Protein Prevents the Production of Porcine Retroviruses , 2003, Journal of Virology.

[4]  Christopher M. Dobson,et al.  A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme , 2003, Nature.

[5]  K. Beam,et al.  Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering. , 2003, Bioconjugate chemistry.

[6]  F. Rippke,et al.  The hair strand test - a new method for testing antifungal effects of antidandruff preparations. , 2003, Journal of cosmetic science.

[7]  C. Verrips,et al.  Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama. , 2002, Journal of dairy science.

[8]  C. Verrips,et al.  Large-scale production of VHH antibody fragments by Saccharomyces cerevisiae , 2002 .

[9]  L. Wyns,et al.  Antigen Specificity and High Affinity Binding Provided by One Single Loop of a Camel Single-domain Antibody* , 2001, The Journal of Biological Chemistry.

[10]  R. Tan,et al.  Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi. , 2001, Journal of biotechnology.

[11]  S. Muyldermans,et al.  Single domain camel antibodies: current status. , 2001, Journal of biotechnology.

[12]  J. Arrese,et al.  Effect of Ketoconazole 1% and 2% Shampoos on Severe Dandruff and Seborrhoeic Dermatitis: Clinical, Squamometric and Mycological Assessments , 2001, Dermatology.

[13]  L. Wyns,et al.  Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. , 2001, Trends in biochemical sciences.

[14]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[15]  C Cambillau,et al.  Thermal unfolding of a llama antibody fragment: a two-state reversible process. , 2001, Biochemistry.

[16]  J. W. Bos,et al.  Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. , 2000, Journal of biotechnology.

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

[18]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

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

[20]  H R Hoogenboom,et al.  Antibody phage display technology and its applications. , 1998, Immunotechnology : an international journal of immunological engineering.

[21]  J. Boonstra,et al.  Impaired Secretion of a Hydrophobic Cutinase bySaccharomyces cerevisiae Correlates with an Increased Association with Immunoglobulin Heavy-Chain Binding Protein (BiP) , 1998, Applied and Environmental Microbiology.

[22]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[23]  B. Petschow,et al.  Reduction in Virus‐Neutralizing Activity of a Bovine Colostrum Immunoglobulin Concentrate by Gastric Acid and Digestive Enzymes , 1994, Journal of pediatric gastroenterology and nutrition.

[24]  A. Scheynius,et al.  Identification of allergen components of the opportunistic yeast Pityrosporum orbiculare by monoclonal antibodies , 1994, Allergy.

[25]  D. Rosenthal,et al.  A randomized, double-blind, placebo-controlled trial of ketoconazole 2% shampoo versus selenium sulfide 2.5% shampoo in the treatment of moderate to severe dandruff. , 1993, Journal of the American Academy of Dermatology.

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

[27]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[28]  G Vriend,et al.  The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease. , 1992, Protein engineering.

[29]  H R Hoogenboom,et al.  By-passing immunization. Human antibodies from V-gene libraries displayed on phage. , 1991, Journal of molecular biology.

[30]  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.

[31]  R. Sauer,et al.  The structural stability of a protein is an important determinant of its proteolytic susceptibility in Escherichia coli. , 1989, The Journal of biological chemistry.

[32]  S. Ho,et al.  Site-directed mutagenesis by overlap extension using the polymerase chain reaction. , 1989, Gene.

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

[34]  S. Shuster.  THE AETIOLOGY OF DANDRUFF AND THE MODE OF ACTION OF THERAPEUTIC AGENTS , 1984, The British journal of dermatology.

[35]  G. Priestley,et al.  The microbiology of dandruff , 1976, The British journal of dermatology.

[36]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[37]  G. Piérard,et al.  Correlation between Malassezia spp. load and dandruff severity , 1998 .

[38]  A. Schmidt.  Malassezia furfur: a fungus belonging to the physiological skin flora and its relevance in skin disorders. , 1997, Cutis.

[39]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[40]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[41]  E. Kabat,et al.  Sequences of proteins of immunological interest , 1991 .

[42]  B. Bokhout,et al.  The influence of a water-in-oil emulsion on humoral immunity. , 1986, Comparative immunology, microbiology and infectious diseases.

[43]  K. Hechemy,et al.  A Comparison of the Bacterial and Yeast Flora of the Human Scalp and Their Effect Upon Dandruff Production , 1967 .