STUDIES ON SOME IMPORTANT PROTEINS AND PEPTIDES FROM PLANT AND MICROBIAL SOURCES

Abstract

Proteins are important functional elements of the cell, present in all kinds of life forms ranging from viruses to eukaryotic cells. Within, they perform various biochemical functions such as hormones, enzymes, antibodies, blood coagulating factors, receptors, signaling molecules, et cetera. They are also known to perform various structural and mechanical roles as keratin of hairs, nails and actin and myosin fibre bundles in the muscles, respectively. The chief characteristic of the proteins that enables them to carry out their diverse cellular functions is their ability to bind and interact with other molecules specifically and tightly. This binding ability of the protein is greatly influenced bythe three-dimensional structure of the protein molecule, it's surface topology, the nature of the amino acids exposed on the surface or involved in the interaction with the other molecules and the physical and chemical properties of the surrounding medium in which the interactions of the protein molecule with the other molecule takes place. Formation of specific complexes between antigen and antibody is a classic example of high complementarity and specific interactions of protein molecules with various other molecules. These interactions are completely dependent on the structural and functional stability of the proteins in different physicochemical conditions. The structural and functional characteristics of the proteins can be examined both in-vitro and in-vivo conditions. In order to perform in-vitro analysis, a protein must be purified from other cellular components. The process usually involves cell lysis, centrifugation and various chromatographic methods. Purified protein can be further characterized biochemically by various bioassays and biophysically by using various biophysical techniques like UV-Visible Spectroscopy, Circular Dichroism, Fluorescence Spectroscopy, X-ray crystallography and NMR. This thesis is divided into four chapters and covers the studies carried out on three important classes of proteins, which are: procaryotic respiratory nitrate reductases, an antimicrobial protein and a protease inhibitory peptide. Chapter 1 provides the literature review, briefly describing the various types of nitrate reductases, plant proteinase inhibitors and antimicrobial peptides found in the nature. It also covers briefly the various roles of protease inhibitors and antimicrobial peptides, with emphasis on their mechanism of action. Chapter 2 describes the cloning and sequencing of the p-subunit of procaryotic respiratory nitrate reductase gene from Bacillus circulans NCIM 2107. PCR primers used for amplification of beta subunit of respiratory nitrate reductase were designed from different strains of Bacillus such as Bacillus licheniformis, Bacillus lentus, Bacillus pumilus and Bacillus subtilis. The sequence obtained directly with the transformed E. coli DH5a cells containing the recombinant plasmid consisted of 729 bases which included the part of the nitrate reductase P-subunit gene containing an Open Reading Frame (ORF) of 678 bases. The ORF translates into a part of P-subunit of respiratory nitrate reductases containing 226 amino acids. Homology model of the part of translated p-subunit of respiratory nitrate reductase was obtained using the modeler program. Chapter 3 covers the purification of a peptide with trypsin inhibitory activity as well as antimicrobial activity from the seeds of Lagenaria siceraria (Bottle Gourd) by cation exchange chromatography on CM Macroprep column and followed by reverse phase chromatography on HPLC with retention time of 3.848 minutes, eluted at 25.6% acetonitrile concentration. The molecular mass of the protein determined by MALDI-TOF analysis was found to be 678.9 daltons. The molecule purified was confirmed to be a peptide by FTIR spectroscopic studies with the peptide giving the infra-red absorbance of 1639 cm" , which is characteristic of the amide bond. The peptidic nature of the compound was further confirmed by the strong absorption peak of the molecule in spectrophotometer at 214 nm, which is the characteristic absorbance for the amide bond in ultraviolet region. It was further investigated that the purified compound completely inhibited bovine pancreatic trypsin at a molar ratio of 1:2. At the molar ratio of 1:1 the peptide inhibition of bovine pancreatic trypsin was only 57.5%. The purified peptide showed antimicrobial activity against the Escherichia coli with Minimum Inhibitory Concentration (MIC) of 20 uM. Antimicrobial activity was also exhibited by the seed extract of Lagenaria siceraria against Escherichia coli when 35 ul of the extract was used for inhibition of bacterial growth. Chapter 4 describes the purification of another antimicrobial protein from the seeds of Citrullus fistulosus (Indian round gourd or Tinda) by two step purification method involving first anion exchange chromatography on DEAE Macroprep followed by cation exchange chromatography on CM Macroprep. The molecular weight of the purified peptide determined by SDS-PAGE analysis under reducing conditions was found to be 9 kD. Efforts for desalting of the protein fractions and to concentrate them using centricon and centriprep of low molecular weight cutoffs were futile as no protein was detected in the filtrate thus desalting and further concentration of the protein fractions was not carried out. N-terminal sequencing of the first seven amino acids of the protein was carried out by Edman degradation. TheN-terminal amino acid sequence of the first seven amino acids was Phe-Asp-Asn-Ser-Phe-Thr-Asn or FDNSFTN and showed no identity or similarity whatsoever to any class of protein from the non-redundant protein sequence (nr) database. The purified protein showed significant antimicrobial activity against Staphylococcus aureus.