recombinant protein

Examples of recombinant protein in the following topics:

• Purifying Proteins by Affinity Tag

  • Protein tags are peptide sequences genetically grafted onto a recombinant protein.
  • Protein tags are peptide sequences genetically grafted onto a recombinant protein.
  • These include chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).
  • Solubilization tags are used, especially for recombinant proteins expressed in chaperone-deficient species such as E. coli, so as to assist in the proper folding in proteins and keep them from precipitating.
  • BCCP (Biotin Carboxyl Carrier Protein), a protein domain recognized by streptavidin

• Crystallographic Analysis

  • Nuclear magnetic resonance spectroscopy and x-ray crystallography have become the methods of choice for understanding three-dimensional protein structures.
  • Electron crystallography has been used to determine some protein structures, most notably membrane proteins and viral capsids.
  • Studies of protein crystallography help determine the three dimensional structure of proteins and analyze their function alone or within multimolecular assemblies.
  • The protocol for completing a successful crystallographic analysis requires production of proteins (cloning, mutagenesis, bacterial culture, etc.), purification of recombinant proteins (such as chromatography of affinity and gel filtration), enzymatic tests and inhibition measurement (spectrophotometry), crystallization, x-rays crystallography and structural analysis, interactions determination (microcalorimetry, fluorescence, BIAcore), conformational analyses (circular dichroism, ultracentrifugation, light scattering), modifications analysis (mass spectrometry), bioinformatics, and molecular modelisation.
  • The Protein Data Bank (PDB) is a freely accessible repository documenting the structures of proteins and other biological macromolecules.

• Biochemical Products of Recombinant DNA Technology

  • Many practical applications of recombinant DNA are found in human and veterinary medicine, in agriculture, and in bioengineering.
  • Recombinant DNA technology is the latest biochemical analysis that came about to satisfy the need for specific DNA segments.
  • Recombinant DNA technology engineers microbial cells for producing foreign proteins, and its success solely depends on the precise reading of equivalent genes made with the help of bacterial cell machinery.
  • Some of the recent advances made possible by recombinant DNA technology are:
  • Isolating proteins in large quantities: many recombinant products are now available, including follicle stimulating hormone (FSH), Follistim AQ vial, growth hormone, insulin and some other proteins.

• Mammalian Proteins and Products

  • Recombinant DNA technology not only allows therapeutic proteins to be produced on a large scale but using the same methodology protein molecules may be purposefully engineered.
  • Genetic modifications introduced to a protein have many advantages over chemical modifications.
  • Bacterial expression systems, due to their simplicity, are often not able to produce a recombinant human protein identical to the naturally occurring wild type.
  • Many mammalian proteins are produced by genetic engineering.
  • Recombinant clotting factors have eliminated this problem.

• Iron-Binding Proteins

  • Iron binding proteins of the innate immune system include lactoferrin and transferrins.
  • Iron-binding proteins are proteins generally used to play roles in metabolism.
  • Iron-binding proteins are serum proteins, found in the blood, and as their name suggests, are used to bind and transport iron.
  • Richardson diagram of recombinant human lactoferrin.
  • Based on PDB (Protein Data Bank) 1b0l

• DNA Mobility Shifts

  • A mobility shift assay is electrophoretic separation of a protein-DNA or protein-RNA mixture on a polyacrylamide or agarose gel for a short period.
  • However, assuming that the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile, complex of nucleic acid probe bound to protein, which is "shifted" up on the gel (since it has moved more slowly).
  • This stability is in part due to the low ionic strength of the buffer, but also due to a "caging effect"; the protein, surrounded by the gel matrix, is unable to diffuse away from the probe before they recombine.
  • If the starting concentrations of protein and probe are known, and if the stoichiometry of the complex is known, the apparent affinity of the protein for the nucleic acid sequence may be determined.
  • This method is referred to as a supershift assay, and is used to unambiguously identify a protein present in the protein-nucleic acid complex.

• Antibody Genes and Diversity

  • The first stage is called somatic, or V(D)J, which stands for variable, diverse, and joining regions recombination.
  • It is due to this randomness in choosing different genes that it is able to diversely encode proteins to match antigens.
  • The second stage of recombination occurs after the B cell is activated by an antigen.
  • Affinity maturation occurs after V(D)J recombination, and is dependent on help from helper T cells.
  • Outline the two stages which result in antibody diversity: somatic (V(D)J) and recombination stages

• Selection

  • DNA recombination has been used to create gene replacements, deletions, insertions, inversions.
  • Gene cloning and gene/protein tagging is also common.
  • Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules.
  • This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA.
  • Thus, both the resulting bacterial population, and the recombinant DNA molecule, are commonly referred to as "clones".

• Generalized Recombination and RecA

  • In homologous recombination, a type of genetic recombination, nucleotide sequences are exchanged between two similar molecules of DNA.
  • Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA.
  • Homologous recombination is a major DNA repair process in bacteria.
  • Recognition of the Chi site also changes the RecBCD enzyme so that it cuts the DNA strand with Chi and begins loading multiple RecA proteins onto the single-stranded DNA with the newly generated 3' end.
  • This type of resolution produces only one type of recombinant (non-reciprocal).

• Genetically Engineered Vaccines

  • For example, this gene may be programmed to produce an antiviral protein.
  • When the chromosome is once again sealed, the bacteria is now effectively re-programmed to replicate this new antiviral protein.
  • Although recombinant subunit vaccines hold great promise, they do present some potential limitations.
  • Recombinant subunit vaccines may also suffer from being too well-defined, because they are composed of a single antigen.
  • This problem can be minimized, where necessary, by creating recombinant vaccines that are composed of multiple antigens from the same pathogen.