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Development of SRMs for Serum Proteomics

Summary:

Serum proteomics, the detection and measurement of as many proteins in a serum sample as possible, could be an important element of personalized medicine enabling diagnosis of early stages of disease. To accurately detect and measure proteins in a biological sample, standards are needed with a sampling of representative proteins. NIST is developing a Standard Reference Material (SRMs) for serum proteomics. An important component of the SRM will be human serum albumin (HSA), the major protein constituent in blood.

Description:

Human serum albumin (HSA) is the major protein constituent of plasma of the blood. The primary role of albumin is to maintain normal osmolarity in the bloodstream. HSA has an exceptional binding capacity for numerous small molecules and hence is an in-valuable transporter of the molecules. HSA is used clinically in multi-gram quantities in applications that include stabilizing blood volume during surgery and cases of shock or burns and in various other clinical situations. Quantitatively, HSA is the world's most commonly used intravenous protein, estimated at around 500 metric tons per annum. Since blood is the major source for the fractionation and purification of HSA, the very large amounts of albumin required demand even larger collection amounts of blood. The use of HSA extracted from human blood supplies is complicated by the possibility of infection with HIV or other viruses and agents. Therefore, there is pressure worldwide to develop alternatives to using donor blood to obtain pure HSA. This work describes experimental production of HSA, engineered for production in E. coli. The HSA pro-duced as part of this work will be used in a SRM for serum proteomics.


Objective

Conduct research to anticipate long-term, short-term, and current metrology needs for serum proteomics projects in government, academia, and industry.


Goals

  • Demonstrate expression of recombinant human serum albumin (HSA) in an in vivo expression system (bacterial or yeast).
  • If necessary, optimize DNA expression vector.
  • If necessary, obtain commercial yeast strain expressing HSA.
  • Demonstrate successful purification of HSA from a recombinant expression system.
  • Demonstrate stable-isotope labeling of HSA in vivo utilizing N15-labeling culture me-dia (bacterial or yeast)


Research activities and technical approach

To express recombinant HSA in E. coli, the gene for HSA was amplified by poly-merase chain reaction (PCR) and cloned into several protein expression vectors. After cloning, sequencing of a recombinant clone was performed to check for the authenticity of the gene.

Initial efforts were made to take advantage of the large number of disulfide bonds of HSA by using an expression vector for fusion protein expression and adding the gene for disulphide bond formation to the HSA gene. Upon gene expression, the cloned gene product would be preferentially transferred to the periplasmic space, a non-reducing en-vironment that would facilitate formation of disulfide bonds in HSA. Although expression of the protein was successful, purification was not, perhaps due to the location of the histidine tag in the middle of the fusion protein making it inaccessible to the nickel resin used for extraction.

The second effort involved cloning the HSA gene into two different expression vec-tors, one of which would produce HSA as a native protein and the other that would pro-duce a fusion protein with a histidine tag at the amino terminus of HSA. Expression of HSA was not observed in either of these clones.

Successful production of HSA was achieved in a heat inducible protein expression vector inserted in an appropriate E. coli strain. HSA expression was observed in this system, as judged on SDS-PAGE by the appearance of a protein band corresponding to the molecular mass of HSA.

E. coli cannot express glycosylated HSA, so future work will involve cloning HSA into yeast for production of glycosylated HSA.

Major Accomplishments:

 

• Successful production of HSA was achieved in a heat inducible protein expression vector

 

Start Date:

October 1, 2007

End Date:

Ongoing

Lead Organizational Unit:

mml

Facilities/Tools Used:

Thermal cycler, DNA sequencer, centrifuges, spectrophotometers

Staff:

Johanna Camara
301-975-4672
johanna.camara@nist.gov

Prasad T. Reddy
301-975-4871
prasad.reddy@nist.gov

David  Bunk
301-975-5071
david.bunk@nist.gov

Contact

Prasad T. Reddy
301-975-4871
prasad.reddy@nist.gov

David Bunk
301-975-5071
david.bunk@nist.gov