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250.253 Protein Engineering and Biochemistry Laboratory
Instructor:
Meeting Times:
Classroom:
TA:
Grades:
Credits:
Office hours:
Textbooks
Prerequisites:
Bertrand García Moreno ([email protected])
Monday, Wednesday, & Friday, 1:00 – 5:30 pm
Biophysics Lab in the UTL, room G89
Meredith Peck ([email protected])
Lab reports, quizzes, pre-lab, and participation/etiquette
4
By appointment
None
None
#1
May 23
Protein structure and function
Introduction to the world of proteins using the PyMol molecular
graphics software. Basic laboratory technique I: pipetting and use
of microbalances
#2
May 25
Protein evolution: from genes to proteins to disease
Central dogma of biology and primer on molecular evolution based
on alignment of sequences. Basic laboratory technique II: UV-VIS
spectrophotometry to measure concentration
#3
May 27
Formulation of hypothesis
In silico mutagenesis of staphylococcal nuclease (SNase) and
formulation of hypotheses. Basic laboratory technique III:
preparation of buffer solutions and determination of pKa values.
#4
June 1
Site-directed mutagenesis
Design of primers and application of the polymerase chain reaction
(PCR) to modify a single amino acid in SNase.
#5
June 3
Bacterial transformation
Agarose gel electrophoresis of PCR products to assess results of
PCR. Incorporation of exogenous DNA in E. Coli to create a clone
for production of recombinant proteins.
#6
June 6
DNA purification
Extraction of DNA for determination of sequence of the plasmid
bearing the gene of interest. Measurement of plasmid
concentration and storage of transformed cells.
#7
June 8
Protein production and purification #1
Growth of E. coli and overexpression of variant proteins. Extraction
of recombinant protein and initial purification using centrifugation
and differential solubility.
#8
June 10
Protein production and purification #2
Final purification of recombinant proteins using ion exchange
chromatography and dialysis.
#9
June 13
Cryogenic protein storage and assessment of protein purity
Flash freezing of protein for long term storage. Mass spectrometry
and SDS-PAGE for determination of purity and molecular weight.
Preparation of buffers for experiments in following weeks.
#10
June 15
Assessment of oligomeric state and enzymatic activity
Blue plate assay to determine enzymatic activity and native-PAGE
to determine oligomeric state. Preparation of buffers for
experiments in following weeks.
#11
June 17
Thermal stability and assessment of 2º & 3º structure
Circular dichroism to determine the content of 2º and 3º structure.
Trp fluorescence to monitor the heat unfolding of the protein.
#12
June 20
Stability against acid
pH sensitivity examined with acid denaturation monitored with Trp
fluorescence.
#13
June 22
Thermodynamic stability
Thermodynamic stability measured by chemical denaturation
monitored with Trp fluorescence.
#14
June 24
Evaluation of results and of hypotheses
Results will be discussed, the properties of different variants of
SNase will be compared, and a research poster will be prepared
summarizing the results of this course.