Engineer Engineering

nicholas d. chester, ph.d.

****B Germantown Avenue 617-***-****

Philadelphia, PA 19118 ********@******.***.*******.***

Summary

Protein Biochemist

Scientist with Industrial experience in purifying recombinant human

proteins for drug discovery platforms and structural analysis.

Knowledgeable in the production of protein expression lines using a range

of hosts and vectors. Proficient in generating, purifying and

characterizing polypeptides. Experienced in protein engineering using

molecular biology and biochemistry techniques. Accomplished and tenacious

problem solver with challenging protein targets.

Key competencies include:

> Deliver materials to multiple teams following timelines.

> Display analytical skills and technical knowledge.

> Introduce new technologies and initiatives.

> Communicate effectively with well designed

presentations.

Professional Experience

Merck Research Laboratories and Schering-Plough Research Institute. West

Point, PA and Cambridge, MA. 2007-2011

Associate Principal Scientist, Protein Science Dept.

. Purified and characterized human recombinant proteins expressed in E.

coli and insect cells:

. Utilized molecular cloning to make expression plasmids. Made

baculovirus to infect insect cells.

. Tested for soluble expression of the recombinant protein under a range

of growth conditions.

. Delivered purified protein targets to the drug discovery platform ALIS

(Automated Ligand Identification System).

. Gained expertise in employing directed evolution to enhance the

solubility of a protein targets.

. Performed protein purification using a broad range of column separation

steps:

. Used Ni-NTA, ion exchange, hydrophobic interaction, affinity and

size exclusion chromatography.

. Purified tagged and untagged proteins using AKTAFPLC and AKTApurifier

liquid chromatography systems.

. Introduced and successfully employed a protein solubility matrix into the

Protein Science Dept.

. Expertise was gained in protein biophysical characterization techniques:

. Employed dynamic light scattering (DLS) and SEC to determine the

oligomeric state of proteins.

. Used mass spectrometry to verify protein identity and find post-

translational modifications.

. Employed circular dichroism (CD) to check for proper polypeptide

folding.

. Performed enzyme activity analysis using the fluorescence quenching

molecular beacon (MB) assay.

. Showed ligand binding using thermal denaturation fluorescence (TdF), and

isothermal calorimetry (ITC).

. Experienced in the use of limited proteolysis to re-engineer recombinant

proteins. Delivery of target proteins to structure groups sometimes

required polypeptides to be reduced in size in order for crystals to

form:

. Performed the technique of limited proteolytic digestion to reduce a full-

length protein in size from 85 kDa to 33 kDa.

. Re-engineered a new plasmid to express only the catalytic core domain.

. Delivered a highly purified preparation of the 33 kDa truncation to a

crystallography group.

. Demonstrated ligand and cofactor binding to the new catalytic core

protein by TdF.

. Employed different hosts to generate difficult to express proteins. Some

human proteins cannot be made in E. coli due to lack of solubility,

oligomeric state or cellular processing requirements:

. Knowledgeable in expressing and purifying recombinant proteins

using baculovirus infection of insect cells.

. Experienced in small volume insect cell culture and large volume

culture with ten liter Wave Bioreactor bags.

. Purified one secreted recombinant protein from CHO cell media.

harvard medical school, Boston MA 1996-2007

Postdoctoral fellow, Laboratory of Dr. Phil Leder, Department of Genetics

. Used gene targeting in mouse embryonic stem cells to make a mouse model

for the human disease Bloom's syndrome:

. Analysis of the Bloom's (Blm) mutant embryo and Cre-loxP conditional

knockout Blm mouse yielded four papers.

. Determined a cause of embryo death employing pathology analysis on

whole and sectioned Blm mutant embryos.

. Raised and purified antibodies to the mouse Blm helicase to determine

allele specific protein level differences.

. Generated immortal tumor cell lines to assay for long-term

consequences of Blm mutant DNA damage.

. Gained experience in molecular biology, cell biology and in vivo mutant

mouse analysis:

. Experienced at gene cloning and mapping, plasmid construction with

cDNAs and mammalian cell transfection.

. Proficient at histochemistry and immunohistochemistry of cultured

cells and fixed mouse tissues.

. Expertise in western blotting, RNA and DNA blotting, phospho-protein

analysis, quantitative immunoprecipitations.

. Experienced in mouse primary cell culture. Skilled at generation of

immortal cell lines from mouse neoplasms.

Cold Spring Harbor Laboratory, Cold Spring Harbor NY 1990-1995

Graduate Student, Laboratory of Dr. Daniel Marshak

. Developed an optimization assay to facilitate generation of specific

PCR products. This resulted in a first author paper.

. Performed biochemical analysis on a kinase (CKII) in human cells

(HeLa). Learned a range of biochemical techniques:

. Production of subnunit specific polyclonal antibodies.

. Kinase activity (Lineweaver-Burk) assays.

. Subcellular fractionation, radiolabeling of cells, cell cycle

synchronization, western blot and immunoprecipitation.

. Discovery and characterization of CKII isoforms led to a publication

and a Ph.D. dissertation.

Graduate Student, Laboratory of Dr Michael Wigler (1989)

. Made ras gene mutants employing recombinant DNA techniques. The work

resulted in a publication authorship.

Chiron Corporation, Emeryville, CA 1985-1988

Technician

. Expressed Factor VIII coagulation factor in mammalian cells. Assayed for

expression in transiently or stably transfected cells using ELISA.

Selected for highest expressing CHO mammalian cell clones and expanded

them for production.

. Purified untagged recombinant HIV proteins from yeast cells.

Teaching and

Supervision

. Taught lower division biology and upper division biochemistry laboratory

courses at SUNY Stony Brook for two years.

. Supervised two summer graduate students and two part time technicians

while in Phil Leder's laboratory at Harvard.

Education

Ph.D., Molecular Biology and Biochemistry, SUNY Stony Brook, Stony Brook,

NY

Thesis advisor, Dr. Daniel Marshak.

A.B., Molecular Biology, UC Berkeley, Berkeley CA

Awards

1997-2000. Research fellow, Leukemia Society of America/The Leukemia and

Lymphoma Society.

2010. Received Shining Points Award from Schering Plough Research

Organization.

Publications

1. H Babbe, N Chester, P Leder and B Reizis. 2007. The Bloom's syndrome

helicase is critical for early thymic development of T lymphocytes. Mol

Cell Biol. 2007. 27: 1947-1959.

2. N Chester, H Babbe, J Pinkas, C Manning and P Leder. 2006. Mutation of

the murine Bloom's syndrome gene produces global genome destabilization.

Mol Cell Biol. 2006. 26:6713-6726. Cover art photograph published in

February 2007, vol. 27 issue 3.

3. LD McDaniel+, N Chester, M Watson, AD Borowsky, P Leder, and R Schultz.

Chromosome instability and tumor predisposition inversely correlate with

BLM protein levels. DNA Repair 2003. 2:13807-1404. +N Chester and LD

McDaniel are co-authors.

4. N Chester, F Kuo, C Kozak, CD O'Hara and P Leder. Stage-specific

apoptosis, developmental delay and embryonic lethality in mice homozygous

for a targeted disruption in the murine Bloom's syndrome gene. Genes

Dev. 1998. 12:3382-93.

5. JA McElhinny, SA Trushin, GD Bren, N Chester and CV Paya. Casein kinase

II phosphorylates I kappa B alpha at S-283, S-289, S-293, and T-291 and

is required for its degradation. Mol Cell Biol. 1996. 16:899-906.

6. N Chester, IJ Yu and DR Marshak. Identification and characterization of

protein kinase CKII isoforms in HeLa cells. Isoform-specific differences

in rates of assembly from catalytic and regulatory subunits. J. Biol.

Chem. 1995. 270:7501-14.

7. N Chester and DR Marshak. Dimethyl sulfoxide-mediated primer Tm

reduction: a method for analyzing the role of renaturation temperature in

the polymerase chain reaction. Anal. Biochem. 1993. 209:284-90.

8. J Colicelli, J. Field, R. Ballester, N Chester, D. Young, and M Wigler.

Mutational mapping of RAS-responsive domains of the Saccharomyces

cerevisiae adenylyl cyclase. Mol Cell Biol. 1990 10:2539-43.

9. IC Bathurst, N Chester, HL Gibson, AF Dennis, KS Steimer, and PJ Barr. N-

myristylation of the human immunodeficiency virus type 1 gag polyprotein

precursor in S. cerevisiae. J. Virol. 1989. 63:3176-9.

In preparation:

1. N Chester, Blair Weig, Guichy Waller and Shane Taremi. SIRT1 Catalytic

Domain Binds Substrate, NAD cofactor and an

Inhibitor. 2013.

Conferences

1. May 2010. PEGS Protein Engineering Conference, Boston, MA. Presented

poster.

2. 2005. Genome Instability and Repair. Keystone Symposia, Taos, NM. Gave

talk in addition to poster presentation.

3. 2002. DNA helicases, cancer and aging. Keystone Symposia, Lake Tahoe,

CA. Presented poster.

4. 1998. Mouse molecular genetics. Cold Spring Harbor meeting, Cold Spring

Harbor, New York. Presented poster.

5. 1996. Mouse molecular genetics. Cold Spring Harbor meeting, Cold Spring

Harbor, New York.

6. 1997. Workshop on Werner syndrome and related helicase diseases. NIH and

National Institute on Aging. Bethesda, MD. Presented talk.

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