“Optimization of Production of Recombinant Alpha A Crystallin”
Abstract
This project aims to produce alpha A crystallin and weigh the final product using light scattering. Alpha A
crystallin is a chaperone protein and is linked to formation of cataract. This project entails knowledge of
physics and biology in that light scattering is a common tool used by physicists which measures the
amount of light scattered at a certain angle, ultimately giving the molecular weight. Purified Alpha A will
be important to have in further research because currently, only cow proteins are used and it is
uncertain as to whether the results are applicable cross species.
The specific goals of this project would be to first produce recombinant Alpha A in E.Coli BL21 and then
vary conditions such as media composition, incubation time and growth conditions to optimally produce
the protein. Isolation of the proteins using methods such as freeze/thaw, sonication and enzymatic
treatments will be tested. Scaling up production will then be done so large scale amounts of proteins
can be used in subsequent labs. Finally, light scattering will be used to determine molecular weight.
Background
The purpose of this project is to produce recombinant alpha a crystallin and study the different types of
interactions it makes with the other crystallin proteins. The alpha a crystallin functions as a chaperone
protein in the vertebrate eye lens and heavily influences the disease known as cataract. During this
disease opacity develops in the crystallin lens, preventing light from passing. The opacity of the lens is
linked to the insolubility or breakdown in function of alpha a. By producing recombinant proteins of
alpha a, the interactions of this protein can be studied, providing further insight as to what goes wrong
during this process.
Alpha crystallin is a polydisperse molecule containing two subunits, alpha A and alpha B. While these
subunits share around 60% of the same sequence, both subunits have different functions. It will be
important to produce the subunits in isolation so they can be first studied individually and then
compared to other proteins. Currently at RIT, research is being done to separate both alpha A and B
subunits from native bovine protein and results from this experiment will assist in a further
understanding of the two subunits.
Project significance
Currently, alpha a crystallins are taken from young calves. While using cow proteins is an option, there
are concerns about whether or not results from the experiment may only pertain to one species and
may not translate across the species barrier. Being able to produce pure recombinant protein will give
better samples to work with and greater confidence in our results
A unique aspect of this project is the cross disciplinary skills and understanding needed to fully
comprehend the scope of this project. While numerous biology related skills will be needed, techniques
such as light scattering will be used to measure the molecular weight of alpha a, requiring a basic
understanding of physics. Other students have been working on light scattering aspects of alpha
proteins and will be accessible to provide valuable information and experience. Subsequent experiments
can be done to understand more complex interactions and students from biology, chemistry, physics
and mathematics fields will be needed
Project Design
Recombinant alpha A will be grown and expressed in E.coli BL21. To determine the optimum expression
of the protein, parameters such as media composition, incubation time and growth conditions will be
explored. These experiments will be conducted in 100ml test volumes. The eventual aim is to produce
large quantities of recombinant protein. Protein production will have to be scaled up and time will be
needed to determine proper scaling up procedures. Methodology to determine the best method for
isolation of alpha a will then be isolated using freeze/ thaw, lysozyme and/or sonication. Protein will be
purified using either size exclusion or ion exchange chromatography. In order to visualize and separate
the proteins, SDS-PAGE will be used. Proteins concentrations will be assayed using UV
spectrophotometry and molecular weight of these proteins will be measured using light scattering.
This project will be divided into four segments.

Alpha A production will encompass optimization of bacterial growth and protein induction.
Parameters including media composition, incubation time and growth conditions will be
explored.

Isolation of proteins from bacterial cells will be investigated. Methods such as freeze/thaw,
sonication and enzymatic treatments will be tested. Further purification by size exclusion
chromatography and ammonium sulfate precipitation will be performed. SDS page will be used
to visualize the resulting proteins.

Scaling up production of alpha A production Protein production on a larger scale will be needed
when subsequent labs are done and making sure the protein grows at the same efficiency at a
larger scale is important.

Light scattering will be used to determine the molecular weight of the proteins isolated
The goal of this project would be to produce alpha a recombinant proteins as efficiently as possible, use
light scattering to determine molecular weight and study interactions of alpha A with other crystallin
proteins. By developing a set procedure of making alpha a recombinant protein, research into means of
production of alpha b, beta and gamma crystallin could be evaluated in the future.