Dr W Ashraf BSc, PhD (Warwick)
Senior Lecturer in Microbiology
Department of Biomedical
Sciences
University of Bradford
BRADFORD
BD7 1DP
UK
Office: H20 Richmond Building
Phone: +44 (0)1274 233589
email W.Ashraf@bradford.ac.uk
Contents
Research
Teaching
Administration
Working with the Community
Working with Biotechnology Companies
Working Overseas
Protein-folding and processing
Proteins control cellular processes through the mediation of enzymes. The key to the cells successful use of proteins depends largely on producing and maintaining their structural integrity via protein folding and/or processing. All cells use a very highly conserved group of proteins, known collectively as molecular chaperones, to assist in protein-folding reactions and the assembly of protein complexes in vivo. There has been much interest in molecular chaperones over the last 5 years as to their roles in fundamental biological processes, health &disease and their biotechnological potential.
The following important questions are being addressed:
How do cells ensure proteins are folded correctly?
What are the biotechnological applications of understanding how proteins fold?
How do cells folded proteins correctly?
All organisms respond in a highly conserved manner to unfavourable conditions such as heat shock and stress (for example exposure to ethanol, heavy metals, amino acid analogues and viral infection), by the vigorous and transient acceleration in the rate of expression of a small number of specific genes. The products of these genes are commonly known as stress or heat shock proteins (hsps). Besides the activation of hsp genes the expression of most other genes is inhibited as a result of stress. Some of these proteins are also synthesised constitutively underlining their importance in normal cellular metabolism. Three main classes of hsps exist in nature with molecular weights of approximately 60kD, 70kD and 90kDa (hence hsp60, hsp70 and hsp90). In E. coli these are represented by the proteins GroEL and DnaK, which were initially described following studies of bacteriophage l E.coli interactions, and HptG respectively.
Members of the hsp70 and hsp60 families are also molecular chaperones which participate in a variety of cellular processes. For example DnaK is involved in protein folding, protein translocation, the assembly/disassembly of protein-protein/-DNA (macromolecular) complexes and the protection of RNA polymerase from heat inactivation. Molecular chaperones assist in the correct non-covalent assembly of other polypeptide containing structures in vivo, but are not components of these assembled structures when they are performing their normal and biological function. GroEL and DnaK also interact with other smaller 'cohort' hsps to form complexes which have been termed 'chaperone machines' which are composed of two or more physically interacting members that work together, in various cellular compartments, to perform their molecular chaperoning functions. For example in E.coli, the hsp70 chaperone machine consists of physical interacting DnaK, DnaJ (hsp40) and GrpE (hsp20).
Hsps are highly conserved at the amino acid sequence level highlighting their evolutionary significance in prokaryotes and eukaryotes. Eukaryotes encode multiple hsp70's homologues which are either constitutive housekeeping heat shock cognates (hscs) or stress proteins.
Our aims of are two fold; they are:
i. to generate, isolate and characterise antibody probes for DnaK/DnaJ and GrpE.
ii. to use such defined immunological probes as tools in structural and functional investigations with respect to :-
(a) DnaK-DnaJ,-GrpE (and hence chaperone machine
formation) and -S 32 interactions and,
(b) the renaturation of heat inactivated luciferase.
What are the biotechnological applications of understanding how proteins fold?
The new era of biotechnology was heralded in the early 80's with the advent of genetic modification techniques which allowed genes from one species to be transferred into another. The potential, yet to be fully realised, for producing valuable protein drugs or biopharmaceuticals on a large scale is enormous. However, high-level production of heterologous proteins in cells frequently results in misfolding and aggregation of proteins into inclusion bodies. Reports have demonstrated that the production of human growth hormone and procollagenase inclusion bodies in E.coli was significantly reduced by the co-overproduction of DnaK.
Can the use of molecular chaperones be further refined and extended to aid in protein-folding, protection from proteolysis, and protein recovery? More generally we wish to investigate how we can improve the down-stream processing of biopharmaceuticals using concepts developed in protein-folding studies, engineering and physical chemistry. [bpd]
Teaching Profile
Year
1 Microbiology I.
Cell Biology.
Study and laboratory skills.
Year
2 Medical
Microbiology.
Molecular Genetics
& Cell Biology (Module Co-ordinator).
Year
3. Gene Expression
& Cellular Communication.
Advanced techniques
& applications in Biomedical Sciences
(Module Co-ordinator).
Prions
Offered Projects in: Microbiology, Biotechnology, Biochemistry
& Molecular Diagnostics
M.Sc./Pg.D. Nature of disease: Medical
Microbiology.
Supplementary Skills: Practical course in gene cloning and gene analysis.
Courses
BSc Biomedical Sciences Course Tutor, 1997--present
BSc Biomedical Sciences Second Year Tutor, 1995--present
Second Year Exams Officer, 1994--1996
MSc Placements Tutor, 1995--1997
Departmental
Member of the Management Advisory Board
Chair Biomedical Sciences Teaching Quality Group (TQG)
Co-ordinator for the Departmental Subject Review (1998-2000)
[Molecular Biosciences]
Deputy Chair of Academic Committee
University
Senate 1996 to 2000---elected member from the Assembly
Deputy Biological Safety Officer---with special responsibility for microbiology and genetic modification
Joint Committee of Senate and Students Union---Member
Microbiological & Genetic Manipulation Sub-Committee---Member
Contact Scientist -Calverley Parkside Primary School, Leeds
Introducing Life Sciences to primary school children including:
Introduction to Microbiology
Interpretation of microbes byAlia, Lianne and Zehra (all aged 8
years old)
Building Cells
Isolation of DNA from plants
Working with Biotechnology Companies
Consultant in Biotechnology for Cytocell Ltd
Dr D. Cardy, Research Director & Honorary Visiting Lecturer in Biomedical Sciences
Cytocell® develops and manufactures products based on innovative,
convenient DNA probe technology for the detection of genetic disease, cancer genes and
infectious agents
The Chromoprobe® range of kits use a proprietary system that simplifies Fluorescence In-Situ Hybridisation (FISH), for the rapid detection of chromosomes. DNA probes are applied to specially treated coverslips using a parented process. This technology reduces the number of steps in the procedure, since denaturation of the probe and target DNA occur simultaneously.
The kits also contain all key reagents optimised to give fast reliable results
Academic collaborator with Bradford Particle Design Ltd (bpd)
Dr G. Humphreys, Managing Director:
BBSRC/DTI LINK Biochemical Engineering Programme.
'Investigation of supercritical fluid technology to produce dry
particulate formulations of proteins'.
P. York (Bradford), M. Hoare (UCL) & W. Ashraf (Bradford).
Bradford Particle Design bpd, a new and exciting company formed in
1995 to develop
and commercialise a "breakthrough" process technology for production of
highly-specified chemical powders, using supercritical fluids. The SEDS [Solution Enhanced
Dispersion by Supercritical fluids] process was initially developed at the University of
Bradford in the Department of Pharmaceutical Technology by Professor Peter York and Dr
Mazen Hanna.
The powder processing industries, in particular the pharmaceutical industry, struggle with "old" technology of crystallisation, drying, milling etc. to produce powders which often suffer from problems of batch-to-batch variation, residual solvent, statically-charged particles etc. etc. etc.
The SEDS process offers a radically new approach which allows the
direct production of highly-specified end-product powders and powder-formulations of
chemicals, in particular pharmaceuticals in a simple one-step process. The primary focus
of the company at present is pharmaceuticals, but the technology can also be applied in
other industrial sectors such as agrochemicals, explosives, speciality/effect chemicals,
photographic emulsions, paint/spray coatings.
Faculty of Biotechnology, University of Gdansk & Medical
School of Gdansk, Poland.
Bradford Co-ordinator Tempus JEP 07191-94 Funds: £270,000
1994-1997 Bradford-Gdansk TEMPUS programme in Biotechnology.
Funds under this scheme were used to set-up an novel Faculty of Biotechnology.
This programme has been successful and has enabled the following exchanges of staff and
students in both directions:
Bradford-Gdansk
Staff
4
Students
2
Gdansk-Bradford
Staff
7
Students
undergraduates 9
postgraduates 2
Max Planck Institute for Biochemistry, Martinsried, Germany
Collaborating with Prof. W.Baumeister in the supervision of Dr V. Pamnani (Full time extra-mural student)
Title:
Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase
homologue from the archaeon
Reference:
Pamnani, V., Tamura, T., Lupas, A., Peters, J., Cejka, Z., Ashraf, W. and Baumeister, W.
(1997). Cloning, sequencing and expression of VAT, a CDC48/p97 ATPase homologue from the
archaeon Thermoplasma acidophilum. FEBS Letts. 404 , 263-268
