Institute of Biological Chemistry

The Institute of Biological Chemistry was established in 1972 at the exhortation of the late Professor Cho-Ho Li, who was the Director of Hormone Research Institute at the University of California, Berkeley. At the time of its inception, the Institute made two important resolutions:

1. As its long term objective, the Institute would devote its primary effort to the study of biologically active proteins. It selected the biochemical characterization of snake venom proteins as the immediate short range objective, since Taiwan has had a long history of successful research in this area and was already renowned throughout the world for its pioneering work on a number of venom toxins.
2. To locate the Institute on the campus of National Taiwan University to cross-fertilize the teaching and basic research with the Graduate Institute of Biochemical Sciences.

Building in NTU Campus

Agreement between Academia Sinica and National Taiwan University was signed in February 1973, and a four-story building for the Institute was constructed on the University campus in April 1977. The Institute formally commenced its operations in July of the same year with a handful of researchers. In the following years, the number of research personnel steadily increased to include both postdoctoral fellows and graduate students. At the same time, the subjects of research have greatly expanded and diversified to move beyond the original scope of protein biochemistry into the area of molecular and cellular biology.

In 1995, a new chapter of the Institute was inaugurated with the completion of a new eight-story building on the Academy campus in Nankang to accommodate new research programs initiated by young staff and newly recruited staff. This new building also accommodate the Life Sciences Library (two floors) to be shared by the five life sciences Institutes on the Academy campus. At present, the personnel of the Institute are evenly divided between the two campuses. With the opening of a new highway, the travelling time between the two campuses has been shortened to about 20 minutes.

RESEARCH

The Institute maintains its basic strength in protein biochemistry, biophysics, and physiology, with snake venom and hormones as specific subjects of investigation. At the same time, the Institute continues to evaluate new areas of research and seek opportunities to contribute to the overall effort of the Academy in promoting the biotechnology program in Taiwan. Areas that are under active pursuit by our researchers include: hormones, enzymes of plants and eyes, enzymes for use in bioorganic synthesis, venom toxins and their membrane receptor proteins, proteins of the reproduction system, crystallins, and transcription factors. In these studies the molecular and cellular approaches to the problem are emphasized as well as biochemical aspects. In 1996, with the expansion of the Institute into the new building, a new laboratory of Molecular and Cellular Glycobiology has been established to study the structure, function, and biosynthesis of glycoproteins and glycolipids. Throughout these studies, the Institute adheres to the basic precepts that include:

• The pursuit of quality and excellence with the suppression of bureaucratic routine.
• Preservation of the autonomy of the senior scientists in selecting their fields of research with deep commitment to promoting mutual collaboration among fellow scientists.

PERSONNEL AND FACILITIES

PERSONNEL

The Institute has 2 distinguished research fellows, 12 research fellows, 2 correspondence research fellows, 1 distinguished visiting professor, 1 visiting professor, 7 associate research fellows, 3 assistant research fellows, 6 post-doctoral fellows, 16 research assistants, 1 assistant, 6 technicians and 4 administrative staff.

FACILITIES

Our major facilities include amino acid analyzer, gas phase protein sequencer, peptide synthesizer, DNA sequ-encer, fluorescence spectrophotometer, ultracentrifuge, high performance liquid chromatography, dynamic light scattering spectrometer, laser Raman spectrophotometer, 400 MHz NMR spectrometer, and other standard biochemical equipment. The library has a collection of over 2,000 monographs, reference books and 124 scientific journals.

MAJOR RESULTS OF RESEARCH

ANIMAL HORMONES

DNA Synthesizer

• The signal peptide effect on the secretion of the a and b subunits of carp gonadotropin in the recombinant baculovirus expression system has been compared. This will help produce more gonadotropins in the insect cells.
• The biological activity of carp gonadotropin may be associated with the sulfate groups present in b subunit glycans.
• Protein purification and modification of growth hormone, prolactin, and crystallins.
• Previously, our reports suggested that analogues of LHRH stimulate receptor mediated tyrosine phosphatase activity and reversed the growth promotion of the tyrosine kinase class of oncogenes. Our recent results indicated that analogues of LHRH could also inhibit the induction of metalloproteinase in a great spectrum of tumor cell lines. These results suggest that analogues of LHRH might prevent the invasion action of tumor cells.

ANIMAL TOXINS AND RECEPTORS

Liquid Scintilation Counter

• Mastoparan B is a cationic tetradecapeptide isolated from the hornet Vespa basalis venom. The peptide was found to cause cardiovascular inhibition and induce antibody without conjugation to a protein carrier. Studies on its structure-activity relationship revealed that lysine-2 is crucial for the cardiovascular effect, while the lysine residues located at N- and C-terminal segments of the peptide, especially lysine-4, plays a crucial role in the binding interaction and the immunogenicity of mastoparan B.
• Site-directed mutagenesis of Phe-22 to Tyr converted a nontoxic phospholipase A into an inhibitor for the binding of a neurotoxic phospholipase A to synaptic membrane. Other mutants have also been made.
• The phospholipase variants from Taiwan and Fujian Deinagkistrodon acutus snake venom were cloned and their amino acid sequences were determined. The phylogenic trees of the group II phospholipases A were constructed based on more than 60 sequences of the enzymes.
• Site-directed mutagenesis of a neurotoxic phospholi-pase A from Taiwan habu venom successfully identi-fied residues responsible for neurotoxicity, e.g. Asn6 and Leu125.
• The amino acid sequence and function of the heter-odimeric anticoagulant proteins from Echis carinatus leucogaster venom were analyzed.
• The tertiary structure of mastoparan B, waglerin I and des(46-49)-[Ala^8,37]-echistatin g has been studied by NMR, distance geometry and restrained molecular dynamics.
• Developed a rapid synthesis method to prepare snake toxins and analogs (62-residues) within two weeks by chemical synthesis, and best folding conditions were determined.
• Partial amino acid sequences of a binding protein in the synaptic membrane of porcine brain for a neurotoxic phospholipase A have been determined.

BIOORGANIC AND STRUCTURAL CHEMISTRY

g Counter

• Isolation of new thermo-stable lipase and protease for high temperature catalysis in organic synthesis.
• Engineering of protease inhibitors and determining its 3-D structure.
• Developed an enzymatic procedure to resolve chiral drug intermediate homophenylalain by lipase.
• Development of a method for studying physical and functional interaction of proteins. Protein complex is isolated by chromatography, immunoprecipitation, or protein-protein interacting procedures. The purified protein (complex) is then subjected to LC/MS/MS analysis after proteolytic digestion. The identity of the protein is obtained by searching the sequence homology in the data bank.
• Developed a microwave technology in peptide synthesis, peptide bond hydrolysis, glycosylic bond hydrolysis and enzyme-catalyzed synthesis of peptides and glycopep-tides.
• DNA autosequencing.

REPRODUCTION SYSTEM

Amino Acid Sequencers

• A sperm motility inhibitor (SMI) has been purified from porcine seminal plasma and characterized. SMI was found to be identical to a mucus-associated protein, the beta-microseminoprotein, on the basis of sequence analysis and mass determination by electrospray ioni-zation mass spectrometry. Furthermore, SMI was found to be an inhibitor of Na⁺, K⁺-ATPase. These results are useful clues for the elucidation of the in vivo functions of this wide-spread protein.
• A glycoprotein in mouse uterine luminal fluid was purified to homogeneity via a series of steps. Auto-mated Edman degradation was unable to determine the N-terminal residue of the glycoprotein, and the partial sequences determined were found to be identical with the protein sequence deduced from 24p3 cDNA. Results of Northern-blot analysis for various tissues of adult mice revealed that the 24p3 gene was expressed in lung, spleen, uterus, vagina and epididymis.
• The carp cystatin has been purified from ovarian fluid. Its cDNA was also cloned and sequenced.
• Carp ovarian cDNA library was constructed from which several types of cDNA coding for egg membrane components, such as ZP2 and ZP3, were isolated. The genomic structure and expression of carp ZP3 were studied.
• The genomic structure of SVA (Seminal vesicle auto-antigen) was determined. Based on the sequence of 5'-flanking region, the functional assay for the androgen response element was attempted. Moreover, we demonstrated specific binding of SVA to sperm by histochemical study and binding assay.

TRANSCRIPTION FACTORS AND OTHERS

Ultra Centrifuge

• Expression of the alpha-1 acid glycoprotein gene (agp) is regulated by AGP/EBP in liver during the acute phase response. However, the molecular mechanism for this regulation is poorly understood. We have demonstrated that two activator forms of AGP/EBP, one of which has an additional 21 amino acids at its N-terminal region, are expressed in liver as well as in a number of cell lines. We have also demonstrated that NF-kB and the AGP/EBP activators can induce the transcription of agp synergistically in an AGP/EBP-binding motif-dependent manner.
• Identification and characterization of stress signal-induced transcription factors involved in the regulation of agp/ebp gene transcription were made. Specifically, c-Jun, ATF2, and NF-kB were identified to be the factors responsible for the UV- or a number of cytokines-induced transcription of agp/ebp gene.
• We have identified Nopp140 as AGP/EBP interacting protein by immunoaffinity chromatography followed by LC/MS/MS analysis. Nopp140 has also been shown to be a transcription factor, and involved in induction of agp gene in an AGP/EBP-binding motif-dependent manner.
• Expression of angiotensinogen gene was regulated by transcription factors NF-kB and AGP/EBP (C/EBP ) through an acute phase responsive element (APRE) in angiotensinogen promoter. These two transcription factors activate the angiotensinogen promoter syner-gistically.

  Peptide Synthesizer

• The genomic structure and promoter region of carp JAK1 kinase gene have been determined and charac-terized. We will further investigate the control mechanism in the transcriptional regulation of JAK1 kinase gene expression.
• A mannose-binding protein, an important protein relevant to immunity, was purified from bighead carp serum by affinity chromatography and shown to have hemolytic activity.
• Among the factors that favor the formation of amylo-plast in cultured sweet potato cells, osmotic stress has been identified as the dominant factor.

RESEARCH STAFF

MEMBERS OF THE ADVISORY COMMITTEE