Voyages into creating infrastructure for studying Proteins
Prof K Kannan, Vice Chancellor, Nagaland University, Lumami
vicechancellornu@yahoo.com

It is indeed an honour to be able to address an audience who are so young and yet dabbling with advances in technology and its applications. Here an attempt is made to take one through four decades of attempts at sequencing proteins in the country.

Introduction:
Meeting my cousin Dr R M Iyer of BARC in the year 1964 moulded my decision to pursue a career in science. The chance introduction by Prof V.V.S. Murthy in the year 1970 (Chemistry department, Delhi University) of the book on “Structure and Action of Proteins by R.E.Dickerson and I. Geis with colourful illustrations deepened my love for the subject of proteins. Not only that, Prof Murthy allowed me to work in his laboratory during my free time. He demonstrated to me the working of 60MHz NMR machine, IR, UV spectrometers and how one purifies amino acids from natural products using preparative paper chromatography and column chromatography. This made me realise the total dependence on analytical techniques based instrumentation if one wants to advance frontiers of knowledge through reasoning, visualisation, predicting and validating the likelihood of how nature operates. Scientist like Dr Jagadish Chandra Bose, Sir C V Raman, Prof P T Narasimhan and Dr R M Iyer  built  their own instruments specifically to answer their ideas, reason it out  and demonstrate experimentally. The establishments such as Atomic Energy program, Space program and Defence Program have fabricated their own instruments and become self reliant, probably due to ban on imports by them.
Looking back to 70’s, we had never sequenced any protein. Notable contribution of Prof G N Ramachandran on various aspects of 3-D structure of collagen in 1954 and prediction of secondary structure through Ramachandran plot in 1967 were indeed a silver lining in Protein research. There are indeed several unsung heroes like Dr D Rajagopal Rao, Prof Paul J Vidhyathil, Prof L K Ramachandran, Prof A. N Radhakrishnan, Dr Narasinga Rao, Prof M Vijayan, Prof P Balram, Prof Salahudhin among many others who sincerely made efforts to create laboratories to study chemistry of proteins in the country. 
Donation of Beckmann analytical Ultra centrifuge and amino acid analyzers in 50’s to few institutions such as NCL Poona, CFTRI, Mysore, RRL, Hyderabad( later transferred to CCMB) and Delhi University gave confidence to attempt to do good work on biophysical and biochemical aspects of proteins. To my knowledge analytical ultracentrifuge and many complex analytical instruments  are  even now fully functional at CFTRI, Mysore and CCMB. This would not have been possible without the commitment and dedication of instrument engineering division headed by Mr Venkat Kuppaiah at CFTRI, Mr B.Gopinath and now by Mr Shekhar Ramachandran at CCMB.I have hardly seen anyone acknowledging them in their research publication.
History of analytical instrumentation for Proteins:
Any analytical method’s popularity depend on sensitivity and specificity. For example phosphate estimation developed by Y SubbaRow in 1926 was popular till 1980 and has been cited more than 17,000 times. Kjeldalh’s nitrogen estimation has survived more than a century.  Any analytical instrument’s sensitivity depends on the type of detection system. Schlerian optics was used as the detection system in analytical ultracentrifuge fabricated by Svedberg to see macromolecules. His student Tiselius also used the same optics when he fabricated electrophoresis. Development of Poly Acrylamide Gel Electrophoresis(PAGE) in 1964 made Tiselius electrophoresis obsolete. One dimensional PAGE with or without urea or SDS  became routine analytical technique to characterise not only homogeneity of proteins but also helped in characterizing complex proteins with regard to number of subunits.  The development of 2-D Gels became a powerful tool in 1975 to identify over 1500 proteins from cell extract of E.Coli. This heralded the proteomics field. 
Amino acid analysers were developed and automated by Stein, Moore and Spackman, they used colorimetric method, as ninhydrin gave specific Ruheman purple colour with amino acid when heated to 105 degrees. The colour  could be readily monitored at fixed wavelengths of 570 and 440 nm and recorded. The sensitivity was at microgram level. The technology of instrumentation varied depending on whether it is the Scandinavian group or USA group or the British and European  group to name a few. When it came to sequencing proteins, the British group preferred paper chromatography and electrophoresis, the European group preferred TLC and electrophoresis, USA group preferred ion exchange for separation and purification of peptides. Instrumentation also developed region wise such as high voltage paper electrophoresis in UK, Thin layer electrophoresis in Europe and automated ion exchange machines with UV or visible spectrophotometers and strip recorders.  The ion exchange led to several new and novel materials and led to FPLC, HPLC with gel filtration, ion exchange, affinity or reverse phase chromatography.
Establishing manual Protein Sequencing facility at CFTRI:
The first protein chemistry laboratory for sequencing was established in the discipline of Biochemistry and Applied Nutrition at CFTRI Mysore in the year 1974-1975.I visited as a  NSTS summer student followed by joining for PhD in the laboratory of Dr D Rajagopal Rao, Asst Director to establish the first manual sequencing laboratory. Due to safety considerations, setting up the facility for High Voltage Electrophoresis  was a challenge and we were given a room facing the veranda in the first floor of the main building. The first  paper electrophoresis experiment of amino acids at high voltage( having two or three fire extinguishers) finished in less than one hour as compared to 16 hours doing descending paper chromatography. Staining with ninhydrin revealed several amino acids. Then we tried protein digest and it revealed several peptides. Based on mobility, one learned to arrive at the molecular weight depending on charge, based on Offord plots.
As a PhD student in mid 70’s, it was quite daunting to characterize kappa casein from buffalo milk which had several post translational modifications. Proving the homogeneity was a challenge in itself. The modifications were sialic acid, galactose, galactosamine and phosphate groups. Developing methods to separate several bands was not easy but achieving it was an exciting and frustrating experience. There were no automated machines and only thing available was automated, programmable fraction collectors from Pharmacia. Pharmacia held monopoly on columns, gel materials, fraction collectors, pumps, detectors etc. Biorad   had monopoly on acrylamide based gel materials materials.
The urge to build our own amino acid analyzer was prompted largely because of the necessity. Amino acid analyzer of Beckman which was in the Biochemistry since 1955 was the first big machine I got familiar with.( The Chemistry department at IIT had huge USSR made analytical instruments then occupying huge space of a room and it is amazing how sleek present day instruments look). One analysis took nearly a day and cleaning the resin another day. The run could be completed if there was no power failure! We purchased all the components from Durham company, USA and assembled our analyzer with fluorescence system as part of the Humboldt grant, a major shift from ninhydrin chemistry.   The run could be completed in less than one hour and results were reproducible and hassle free. Further the sensitivity could be pushed to picomolar to femtomolar levels. Prof B K Bacchawat’s students Dr Tambi Dorai and Dr Bishal came to analyse  1.7 mg the Sialic acid binding protein from Carscinoscorpion rotunda in 1978. The experiments of amino acid analysis, end group determination, finger printing on TLC plates and determining the new end groups of peptides obtained from tryptic digestion and partial sequencing were reproducibly repeated three times in less than two weeks.  The number of experiments we could do and still return 0.7 mg was indeed a major step forward in 1978, the significance of which remained largely unappreciated by the scientific community. The end group determination using Dansyl Chloride and standardizing Edman method of manual sequencing was a great learning curve. Then only I realised why so few scientists worked on the difficult task of sequencing proteins. (Dr Fred Sanger took eight years to sequence Insulin! Dr Sanger used FDNB to label peptides and amino acids in the 40’s, which was replaced by Dansyl Chloride to improve sensitivity by thousand fold by Prof Brian S Hartley in 1964). With the advent of DNA sequencing (again by Fred Sanger) all that has changed and hardly anyone does protein sequencing during the last two decades. Work of Fredrick Sanger, Stein and Moore, Hartley, Edman are all part of the history of science with respect to Protein sequencing. DNA sequencing methods are also undergoing revolutionary changes in the 21st century. Every new technology brings in the way we ask questions, the way we design or modify instruments  etc. I dread to venture to predict what revolution nano technology will bring about. The challenge is how to interpret the huge amount of data being churned out.
Proteins and Biotechnology era:
The Biotechnology boom began in mid seventies thanks to Genetic Engineering and Hybridoma technology. The experience with large scale purification of proteins came handy to express new and novel proteins in bacteria and later purify them. My experience in Imperial college, London came handy as I had attempted to purify Rhodanese from 70 Kg of Bacillus Steorothermophilus in the late 70’s and early 80’s along with several groups in England and USA. Interestingly pilot plant facility at Imperial college was set up by Nobel Laureate of Penicillin fame Sir Ernst Boris Chain inside the Biochemistry department in 1962. During my stay in London, hands on experience of using Edman  sequencer, mass spectrometer and HPLC for studying peptides was the next  step in coping up with advances in instrumentation.
The discovery of Enkephalins and SRS-A in 70’s using mass spectrometer  opened a new window for sequencing. In late 70’s one saw field atom bombardment (FAB) mass spectrometer which started pushing the limits of molecular weight determination from hundreds to thousands. MALDI-TOF, ESI could stretched the limits to much higher magnitudes in the late 90’s thanks to the young instrumentation engineer Tanaka at Shimadzu coroporation in Japan. Advantage of Mass spectrometer was the ability to detect post translational modifications on amino acids in proteins. For eg.  gamma carboxyl group in prothrombin, its role in Vitamin K function and blood clotting in 70’s.There are over 300 amino acid modification of proteins are currently known and they are playing a major role in regulation and Epigenetics.
Setting up the state of the art facility at CCMB:
During 82 to 86 it was a challenge in building state of the art   infrastructure for protein work at the newly established CCMB at Hyderabad. The emergence of automatic Edman sequencer revolutionized sequencing of proteins in the 60’s. Leroy Hood and Hunkapillar developed gas phase sequencing, a partial modification of Edman sequencer in the late 70’s. We were the first to procure the Gas phase sequencer from Applied Biosystems in the world at that point of time. Amino acid analyser from LKB, HPLC from Waters and  FPLC from Pharmacia were the other acquisitions. By 1985 there were five HPLC and one of them was a diode array detector. Those days even pharmaceutical industries did not have so many HPLC’s!
Thanks to liberalisation in 1993, there are hundreds of HPLC’s in each industry be it quality control, in process QC or R&D. Mass spectrometers, gas chromatographs, flame photometers etc are routine instruments in many R&D laboratories. Drug discovery has added a new dimension of  opportunities such as IPR, pre and post clinical trials, quality assurance etc and all these laboratories have machines running 24 hrs, seven days a week, 365 days in a year. Requirement of equipments have made many companies have a turnover over 100 crores per year.
Capacity Building for Biotechnology at Poona University:
I was fortunate to be a faculty as part of the new program of Biotechnology post graduate program at Poona University when it began in 1985-86 in the department of Zoology. This was the earnest attempt to capacity building amongst youth in the emerging area of Biotechnology in the country. Proteins being the main product of Biotechnology industry (Insulin, Erythropoietin, Hepatitis B vaccine), it was a pleasure introducing the youth in the class to large scale protein manufacture, downstream processing. Interestingly first three batches produced over 55 UGC-CSIR NET scholarship holders. I am happy that one of the students Dr Eddy Syed is Director of manufacturing at ( International AIDs Vaccine In initiative (IAVI) in New York and still fondly remembers their training as first batch of MSc Biotech students.
A decade of experience in the industries:
The dabbling with techniques in the industry was indeed a novel experience, be it Span Diagnostics or Ranbaxy. Developing techniques for ELISA or Stem Cells in late 80’s and 90’s and attempts at manufacturing and marketing brought me closer to GMP, GLP and rigours of getting approvals. The country was not ready in the 80’s and first r-DNA Indian product appeared in the market only in 1998.
Starting up integrated BTech/MTech program:
Returning back to academics after more than a decade prompted me to start a five and a half year integrated B.Tech/M.Tech program in Biotechnology at the newly established Indraprastha University in Delhi in 1999. This was followed by Biotech program in some Higher secondary schools under CBSE in 2002. The text book of CBSE had separate chapters on old and latest techniques and instrumentation. The early introduction has inspired several students, one being Miss P Chitra of NCBS who has ventured into cutting edge of research on epigenetics and cancer. Technologies such as Micro array for proteins started arriving at the scene in the 21st century and gene sequencing changed the arena of proteins. Computers have become the mainstay and Bio-informatics has become the new jargon, analysing data’s using different softwares.
Future of Proteins:
Thanks to whole genome sequencing, the way we look at proteins are more holistic rather than as individual entities. How proteins behave inside a cell has led to System Biology as the new jargon. Protein classification has become information based rather than structural and functional. Proteomics has become the buzz word among researchers. Though genetic information lays out the blue prints, proteomics reveals the real actors within a cell and their interactions. All the techniques have advantages (strengths) and disadvantages (weakness) and one needs a battery of tools to analyse the data obtained. Mass spectrometry can give instantaneous picture what is happening in a cell, tissue or organism. The weakness is that the approach does not reveal everything about the nature and mechanics of those interactions. Micro array can provide better data on interactions but needs standardization. Using binary approaches it has been possible to map Caenorhabditis elegans interactome map (5500 protein interactions among 3000 proteins. Bioinformatics software tools are predicting several possibilities but experimental validation is lagging behind. Some are using sets of rules to define relationship between cellular components instead of the more conventional method of defining specific interactions and species using differential equations. In silico studies are also being attempted. The next step is to integrate the dynamics of the information flow in cells.
Multi analyte analysis using microarray, it is possible to simultaneously look for several analytes. Our only limitation is sensitivity and specificity. Even now we still need more than 10 billion molecules of any analyte in the sample being analysed, be it fluorescence or chemi luminescence. Even today our nose or insects antenna can detect few molecules and analytical instruments have to go a long way to match living beings.