Technology

About Nobuyoshi Shimizu,
Professor Emeritus of Keio University,

Fundamentals of genome research.

Professor Shimizu began teaching at Keio University School of Medicine in 1983 and had laboratories both at University of Arizona and Keio University for the next ten years. He traveled between the two schools every month for research and teaching, and acquired research grants for both labs, each pursuing different fields in the competition with other labs. He was able to bring about a lot of remarkable results. These efforts became the foundation that brought him to play an active part in the Human Genome Project upon its start in 1989.

He published 597 papers on integrating various research fields such as genetic mapping, growth factors and their receptors, cancer, gene expression regulation, cell division, genome, disease, database etc.
In 2004, he held a symposium commemorating the 20th anniversary of the Shimizu laboratory and invited overseas researchers who had close relationship with Professor Shimizu. Throughout his twenty-four years career, Professor Shimizu tackled top-notch research, working with many colleagues, until he retired from Keio University in 2007.

After being granted the title of professor emeritus from Keio University, professor Shimizu continued his passion for research by founding the GSP Center, Keio University Advanced Research Laboratory in Tsukuba. Unfortunately, during a special lecture at a conference held in Hamamatsu, he fell down with a cerebral hemorrhage, which forced him to stay at a hospital for 3 months and to spend 2 years for outpatient rehabilitation. Following his recovery, he began applied research using a human type artificial antibody library originally developed by the Shimizu laboratory.

However, in June 2015, Professor Shimizu passed away in the middle of his research.

Nobuyoshi Shimizu

Book:
Human Genome World

Mystery of Human Blueprint has solved here!
The leading expert decoding the chromosome 21 and 22 describes topics from "Beginning of Life and Evolution” to “Gene Manipulation and Regeneration Techniques””Human Evolution and Dinosaur Regeneration”
by introduction of TRC MARK's book

Birth:

1941, born in Osaka

Education:

1965, B.S., Nagoya University, Faculty of Science (Chemistry)

1970, Ph.D., Nagoya University, Institute of Molecular Biology, (Molecular Biology)

Awards:

Sarnoff Prize (1965), Naito Memorial Award (1971), Kroc Award (1977), American Cancer Society Faculty Award (1978), NIH Recognition (1983), Japanese Human Genetics Society Award (2000), Chunichi Culture Merit Award (2003), Minister of Education Award (2004), Keio Gijuku Award (2007)

Employment:

  • 1970,

    Research Associate, Nagoya University, Institute of Molecular Biology

  • 1971,

    Postdoctoral Fellow, University of California San Diego, Dept. of Biology

  • 1974,

    Research Associate, Yale University, Dept. of Biology

  • 1977,

    Professor, University of Arizona, Dept. of Cell and Molecular Biology

  • 1983,

    Professor, Keio University School of Medicine, Dept. of Molecular Biology

  • 2007,

    Professor Emeritus, Keio University
    Honorary Director, Advanced Research Center for Genome Super Power

  • Others:


    Special Guest Professor, Nagahama Bio-University
    Special Visiting Professor, Doshisha University
    Visiting Professor, University of Arizona
    Honored Professor, IiIiu Hatieganu University, Medical and Pharmacy, Romania
    Honored Professor, China Medical School

Titles:

Member of Aeroelectronic Committee, Science and Technology Agency
Member of Grants-in-aid for Scientific Research Committee, Ministry of Education
Member of Science and Technology Committee, Health Science Council, Ministry of Health, Labor and Welfare
Review Board Member, Japan Society for the Promotion of Science
Member of Scholarship Committee, The Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care
Chairman for research grant grantor selection, Life Science Foundation of Japan Council

Research history:

  • 1974:

    Organizer of "Human Genome Mapping Workshop" at Yale University; Presentation of "Shimizu Human Genome"

  • 1977:

    Development of detection & quantitative system for Epidermal Growth Factor (EGF)

  • 1980:

    Construction of gene map & chromosome-specific DNA library

  • 1983:

    Construction of BAC (bacterial artificial chromosome) library of human genomic DNA
    Mapping of disease genes; establishment of separation method of several mega-base long-DNA

  • 1989:

    Successfully separation of chromosome 21 and 22 DNAs;
    Construction of the contig-map and the exon map The contig map means the alignment of overlapping cloned DNAs.

  • 1993:

    Setup of Japan Aqua Genome Society (Genomic study of marine products)

  • 1997:

    Cloning of disease genes

  • 1998:

    Participation in the human genome project (HGP) consortium as an international team leader

  • 1999:

    The first report of HGP, DNA sequence and analysis of chromosome 21
    The second report of HGP, chromosome 22

  • 2000:

    The forth report of HGP, chromosome 8

  • 2003:

    The finished report of HGP

Editorial Board Member of International Journals:

Genomics, Mammalian Genome,Somatic Cell Molecular Genetics
Cytogenetics Cell Genetics, J. Exp. Zoology (Executive Editor)
Methods, J. Human Genetics, Expert Review in Molecular Medicine
In Sight (Science), Encyclopedia of the Human Genome
Research Signpost Cancer Science, Chinese J. Medical Genetics
Human Mutation (Communicating Editor), Gene Therapy and Regulation

Council, Professional Society and Community:

Jpn Soc Gene Diagnosis and Therapy (President, Inspector)
Jpn Assoc for Chromosome and Gene Analysis (Special Advisor)
Jpn Soc Human Genetics (Council)
Jpn Soc Biochemistry (Council)
Jpn Soc Molecular Biology (Council)
Jpn Cancer Society (Council)
Jpn Cell Biology (Council)
Jpn Aquatic Genomics (President)
Jpn DNA Academy (President)
School of Genomics (Principle)
International Society for Aquatic Genomics (President)
Human Genome Organization HUGO (Founding Council)
Human Genome Variation Society (Council)

Books:

Hito no Idengaku (Human Genetics), Tokyo Kagaku-dojin (translater);Idenshi Chiryo Kakumei (Correcting the Code), Nippon TV (translater); DNA Saiensu (DNA Science), Igaku-Shoin (translater); DNA Saiensu Raboratori (DNA Science Laboratory), Igaku-Shoin (translater); Hitogenomu no Bunshi Idengaku (Principles of Medical Genetics), Igaku-Shoin (translater); Nippon no Toppu Rannah Shimizu Nobuyoshi ga Toku Hito “Genomu” Keikaku no Kyo to Jitsu (Virtuality and reality on Human Genome Project – Nobuyoshi Shimizu, a Top Runner in Japan Explains), Bijinesu-sha (author);
Hito no Idenshi Mappingu – Taisaibou Idengaku to Idenshi Kougaku no Shinryoiki (Human Genome Mapping – Somatic Genetics), Kodansha (co-author); Saisentan Repohto Nippon “Hitogenomu Keikaku” no Ima (State-of-the-art Report Japan, “Human Genome Project”, Bijinesu-sha (co-author); Hitogenomu Wahrudo (Human Genome World), PHP Kennkyujyo (author); Hitogenomu=Seimei no Sekkeizu wo Yomu (Human Genome = Reading Blueprint of Life), Iwanami Science Library; Genomu wo Kiwameru (Mastering Human Genome), Kodannsha (author), etc.

Major Achievements

Professor Shimizu began his scientific work in the laboratory of Professor Reiji Okazaki during his senior year at Nagoya University where he learned how hard and powerful the research is. During his graduate school years he discovered two Bacillus subtilis phages and analyzed nucleic acids of living fossils.

In 1971 he went abroad to the United States to continue studying and research at the University of California, San Diego for 3 years and at Yale University for 3 years where he devoted himself to research. His focus was making chromosomal maps of human genes using hybrid cells of human and mouse, publishing many papers. In 1974 the first human gene mapping workshop was held at Yale University. In 1977 he was selected as Associate Professor of University of Arizona where he had his own laboratory for the first time.

In the lab, Professor Shimizu challenged mapping related to cell growth factors, and was successful in mapping of the EGF receptor. Since then, this branch of research had evolved into an association between signal transduction through EGF and carcinogenesis, and had continued for over 40 years.

In 1983 he came back as a professor of Dept. of Molecular Biology at Keio University School of Medicine where he continued his research on cell growth factors, and developed the technique to separate human chromosomes. Then, he proceeded to map and sequence genomic DNA fragments. In 1988, after the establishment of HUGO (Human Genome Organization), the Human Genome project established international partnerships and Professor Shimizu's team was selected as an active member. He built the Keio Strategy and steadily walked toward the goal.

In December 1999 Professor Shimizu succeeded in deciphering human chromosome 22 and published his findings in International Science Journal Nature. He announced he had completed reading of human chromosome 21 in 2000 and chromosome 8 in 2003. The international genome analysis ended tentatively in 2003, but their function had been known only about half then.

Professor Shimizu named these unknown genes as "Kaonashi" and used medaka, killifish, to analyze their roles in ontogeny. Concurrently, he cloned a number of disease-cause and disease-related genes and analyzed their functions. Those genes include DiGeorge syndrome, Cat Eye syndrome, and del22 syndrome on chromosome 22, Down syndrome, autoimmune disease, deafness, and bipolar disorder on chromosome 21, Cohen syndrome and familial epilepsy on chromosome 8, Parkinson’s disease, pemphigus, Immunoglobulin, Glaucoma and so on. Especially, Professor Shimizu named the gene of Parkinson's disease as “Parkin” which became a trigger to clarify the etiology of the causative gene of some neurological diseases.

In March 2007 he retired from Keio University, and in June he established a new laboratory, Genome Super Power (GSP) Center in Tsukuba to continue his research. He also established GSP Enterprise, Inc. and developed an extensive and expansive artificial antibody library, which was independently constructed by Professor Shimizu and his colleagues with the hopes of facilitating research, academic curiosity, and learning.

Our Technology

Our antibody library is a single-chain fragment variable (scFv) antibody-displaying phage library.
The repertoire of antibody genes are more than 13-14th power of 10.
Construction of a new Fab antibody library with dramatic improvement is in progress.

Antibody Production Technology

Realizing abundant library utilizing antibody specificity

Antibodies, called immunoglobulin, are protein weapons (protein) produced in the living body (immune system) to identify and neutralize foreign objects such as pathogenic bacteria and viruses. Those foreign objects, targets of antibodies, that antibodies bind to are called Antigens.

Antibodies are classified into two, based on whether they are made using identical immune cells, monoclonal antibodies, or using several different immune cells, polyclonal antibodies.
One of monoclonal antibodies' features include its homogeneity derived from the fact that they are made by two kinds of unique peptides, "Heavy Chain" and "Light Chain" encoded by single genes.

There are two methods to make antibodies. One is by using animals' physical immunological function, and the other is by using recombinant DNA technology without using animals' body.

One of advantages of our antibody library using the recombinant DNA technology is that it is applicable not only to antigens with possible toxic effects, but also to autoantibodies.With our library, antibody gene fragments obtained from the phage genome by screening can produce various kinds of recombinant antibody proteins.

We have developed the original phage-displayed antibody libraries to obtain various useful antibodies.

Useful in State-of-the-Art Antibody Preparation Development

In the areas of biology and medical science research, antibodies are used as various detection tools by utilizing their specificity. In addition, recombinant antibodies which are recently put in practical application are highly valued, and are used in development of various antibody preparation.

Recombinant Antibodies

Most of mammal produce immunoglobulins which consist of five major classes or isotypes: IgG, IgA, IgM, IgD, and IgE. IgG is the most abundant class of immunoglobulin in the blood plasma which is a yellowish coloured liquid component of blood, and is widely used as tools for research.

IgG has molecular weight of 170kDa and consists of two heavy chain peptides (H chain) and two light chain peptides (L chain), each of which are encoded by different genes. The domains binding to antigens are called VH domain and VL domain respectively.

Recombinant antibodies which consists of these peptides linked together with linker peptides are called single-chain variable fragments (scFv) which have molecular weight of approximately 30kDa. scFv's merits include that it is easy to genetically engineer because it is encoded with single gene due to the fact that its molecular weight is small, approximately a fifth of that of IgG.

Our Antibody Library

Antibody library with vast repertoire comparable to that of human body's lifespan production of antibodies and with advanced screening system that utilizes such repertoire.

I.Diversity of antibody genes
  • i. CDR(Complementarity-Determining-Region, Hypervariable Region)
    • Error-prone PCR
    • CDR shuffling
  • ii. VH-VL shuffling
II. Library Construction & Stablity
  • i. Non-expressing VH/VL sub-library
  • ii. Cre-lox Recombination in vivo
  • iii. Stringent tetracycline-regulated promoter
  • iv. Co-expression vector for periplasmic chaperones

scFv Antibody-displaying Phage Library

Our antibody library is scFv antibody-displaying phage library. It consists of populations of phages that have antibody genes inside which encode scFv antibody protein that fuses on the surface of phages infecting E. coli.

Methodology of our Antibody Library Construction

In order to increase antibody gene repertoire, the hypervariable regions (Complementarity-Determining Regions : CDRs) 1-2 and CDR3 within VH and VL gene fragments were shuffled by PCR-mediated ligation. Additionally, VH and VL gene libraries are made separately and then are combined in E. coli cells to generate more than 13-14th power of 10 repertoire of the antibody genes. Practically, however, 10 to 11-12th power of 10 repertoire of those could only be used in screening due to liquid-handling limitation. Nonetheless, it is said to amount to the whole antibody repertoire that human produces during its lifespan.

We are in the process of constructing a new Fab antibody library and screening system beyond physical limit.

Optimization of Protein Expression from Various Antibody Genes in the Repertoire

The host E. coli strain of our library harbors a plasmid vector expressing seven rare codon tRNAs and multiple periplasmic chaperons. They reduce production bias of antibodies derived from human genes to have a functional role in effective utilization of the huge repertoire.

Utilization of Strictly Regulated Expression System for Antibody Genes

Protein expression library is generally so unstable due to production of unnecessary protein for E. coli cells that its amplification will increase gene-deleted or mutated clones in themselves. To solve the problem, we construct protein-unexpressing, stable VH and VL sub-libraries at first, and then convert them to the screening-ready library. The final scFv library is also stable by utilization of a strictly-regulated tetracycline-inducible synthetic promoter instead of frequently used, but leaky lactose-induced promoter.

Screening Methodology

High affinity antibody clone selection using Octet® system,
molecular interaction measurement device

A basic screening procedure is called "bio-panning" which follows a series of such steps as mixing the antibody library solution with an immobilized antigen, washing to remove unbound phages away from the antigen, and harvesting specific binding phages.

We improve screening efficiency of high affinity-binder antibody-displaying phages by exploiting immortalizing methods and washing tips adjusted to antigen properties. The high affinity-binders are harvested with certainty by cleaving a phage body from the antibody by trypsin digestion. We have noticed that acid or alkaline elution generally used leaves some high-affinity binders on the antigen. E. coli cells infected with the recovered phages are super-infected with helper phage to produce phage proteins and to propagate antibody-displaying phages for the next panning.

Normally, after the 3rd panning, E. coli cells infected with the recovered phages are cloned. And the antibody-displaying phages made from the individual cells are selected on the binding ability to the antigen. We use harvested antibody genes to modify them into IgG antibodies and scFv-Fc fusion antibodies.

We do not apply ELISA (enzyme-linked immunosorbent assay) in which signal intensity does not correlate with antibody affinity, but uses Octet® system, molecular interaction measurement device, so that we can select efficiently high affinity antibody clones.

Our library is applicable to various screening methods such as cross-selection and guided molecular selection method

We take advantage of in vitro screening in the way that we can offer the subtraction screening method that removes such antibodies binding to similar antigens and, on the other hand, the cross-selection method to select binders to the same epitope (antibody binding site on an antigen). We also can introduce a guided molecular selection method to obtain phages binding to at neighboring or close sites of the binding sites of an available or existing antibody.