250.253Protein Engineering and Biochemistry Laboratory
Instructor:Bertrand García Moreno ()
Meeting Times:Monday, Wednesday, & Friday, 1:00 – 5:30 pm
Classroom: Biophysics Lab in the UTL, room G89
TA:Meredith Peck ()
Grades:Lab reports, quizzes, pre-lab, and participation/etiquette
Credits:4
Office hours:By appointment
TextbooksNone
Prerequisites:None
#1May 23Protein structure and function
Introduction to the world of proteins using the PyMol molecular graphics software. Basic laboratory technique I: pipetting and use of microbalances
#2May 25Protein 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
#3May 27Formulation 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.
#4June 1Site-directed mutagenesis
Design of primers and application of the polymerase chain reaction (PCR) to modify a single amino acid in SNase.
#5June 3Bacterial 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.
#6June 6DNA 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.
#7June 8Protein 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.
#8June 10Protein production and purification #2
Final purification of recombinant proteins using ion exchange chromatography and dialysis.
#9June 13Cryogenic 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.
#10June 15Assessment 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.
#11June 17Thermal stability and assessment of 2º3º structure
Circular dichroism to determine the content of 2º and 3º structure. Trp fluorescence to monitor the heat unfolding of the protein.
#12June 20Stability against acid
pH sensitivity examined with acid denaturation monitored with Trp fluorescence.
#13June 22Thermodynamic stability
Thermodynamic stability measured by chemical denaturation monitored with Trp fluorescence.
#14June 24Evaluation 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.