Substrate specificity testing of recombinant Factor VII using phage display technology Research project
Hamilton, Paul T

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In the Hamilton Lab at NCSU, we use randomized and designed peptide phage display libraries to characterize the activity and substrate specificity of proteases.  Novo Nordisk was interested in characterizing the activity and substrate specificity of proteolytic coagulation factors and variants they generated through site-directed mutagenesis and/or chemical modification.  The initial target of the project is Factor VII (FVIIa) with focus on determining the substrate specificity of wild type and several variants.

Factor VII-substrate libraries were constructed with an amino-terminal hexa-histidine sequence used to bind the phage to a Ni-NTA resin followed by a randomized Factor VII-substrate sequence and the carboxyl-terminal domain of M13 gene III. Each phage particle displays a unique, potential Factor VII-substrate sequence. Phage displaying this library of fusion proteins were bound to the Ni-NTA resin, washed to remove unbound phage and then treated with Factor VII.  Phages containing a good Factor VII cleavage site were released from the resin whereas phages with sequences that are resistant to cleavage by Factor VII remain bound to the resin.

The work using FVIIa and phage display libraries has shown a consensus substrate sequence which is slightly different from the published FVIIa substrate site determined by other methods.  Biochemical characterization has shown that the phage-derived consensus sequence is indeed readily cleaved by Factor VIIa.

In the Master of Microbial Biotechnology program, they have participated in 7 semester-long, practicum projects over the last 5 years with Novozymes.

Novozymes

Fall 2016

Microbial Control in Biofuel Fermentations

Franklinton, NC

Novozymes

Fall 2016

Gas Fermentation for CO2 Utilization

Franklinton, NC

Novozymes

Fall 2016

Survey of Direct Fed Microbials in Animal Feed

RTP, NC

Novozymes

Fall 2014

Anaerobic Digester Methane to Energy: A Nationwide Assessment

Franklinton, NC

Novozymes

Fall 2014

Market Research Report for Yeast in Animal Feed Products

Franklinton, NC

Novozymes

Fall 2012

Evaluation of Attached Growth Bioreactors for Potential use in Industrial Enzyme Production

Franklinton, NC

Novozymes

Fall 2012

Regulatory and Market Assessment of Biological Soil Crust Reclamation

Franklinton, NC

 

Region(s)/Country(s):
  Denmark
Dates:
  01/01/2012 - 01/01/2016
Institutional Partner(s):
  Novo Nordisk A/S, Denmark


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