Protein Engineering Protocols - Mycobacteriology research center
Protein Engineering Protocols - Mycobacteriology research center Protein Engineering Protocols - Mycobacteriology research center
Incorporation of Unnatural Amino Acids 293.2.2.2. EXPRESSION AND PURIFICATION OF GFPUV INCORPORATING 6f W1. Grow C600F(DE3) plus pET100GFPuv in 200 mL of M9B1TLW+Kn+Ap.2. At mid-log phase, centrifuge cultures and resuspend pellets in 100 mLM9B1TL95%6fW+Kn+Ap with 1 mM IPTG and continue growth for an additional3 h (see Note 4).3. Spin down cultures and lyse pellets in 3 mL B-PER. Centrifuge the lysate for 30min at 15,000g to clear insoluble material. To the soluble fraction, add 10 mMMgCl 2(from a 1 M stock solution) plus 3 U DNAse, and allow the lysate to incubateat room temperature for 10 min.4. Prepare a 3 mL Ni-NTA column (Novagen) by washing with 15 mL water and9 mL binding buffer.5. Pass the soluble fraction over the Ni-NTA column. Wash the column with 30 mLbinding buffer and 18 mL wash buffer, and elute with 18 mL elution buffer.Collect fractions of 0.5 mL from the column, and analyze by SDS-PAGE andexposure to ultraviolet light (fractions with protein are visibly fluorescent). Poolfractions containing purified protein, and concentrate as described inSubheading 3.2.2.1.3.2.3. Purification of Whole-Cell Protein ExtractsFor incorporation of 4fW:1. Grow the cultures of C600p to saturation in 25 mL of the appropriate media.2. Pellet the cultures by centrifugation, and lyse in 200 µL B-PER.3. Pass 50 µL of this lysate through a Centri-Sep size-exclusion column to removeunincorporated amino acids.Similarly, to analyze the level of discrimination of bacteria against 6fW:1. Grow C600F to saturation in 100 mL of culture on either M9B1TLW+Kn orM9B1TL95%6fW+Kn.2. Pellet and lyse the bacteria in 200 µL B-PER II.3. Pass half of this volume through a Centri-Sep column (see Subheading 3.3.1.1.).3.3. Analysis of Incorporation Levels of Unnatural Amino AcidsIn this section, two methods are discussed for determining the extent ofunnatural amino acid incorporation. Complete hydrolysis followed by HPLCand mass spectrometry (Subheading 3.3.1.1.) or HPLC followed by HPLCunder different conditions (Subheading 3.3.1.2.) can be used for purifiedprotein and for whole-cell protein extracts. However, protease cleavage andfragment analysis (Subheading 3.3.2.) can only be used on purified proteins.3.3.1. Analysis of Purified ProteinsSample results pertaining to Subheadings 3.3.1.1. and 3.3.1.2. are presentedin Table 1.
30 Bacher and EllingtonTable 1Incorporation of 6fW Into ProteinMethod of analysis Subheading Incorporation (%)Whole-cell protein extractHPLC-ESI 3.3.1.1 56.5HPLC-HPLC 3.3.1.2 66.7Purified GFPuvHPLC-ESI 3.3.1.1 68.6Average incorporation 64.0From ref. 4.3.3.1.1. HYDROLYSIS AND HPLC-ESI ANALYSIS OF PROTEINS CONTAININGUNNATURAL AMINO ACIDSAcid hydrolysis of protein samples allows for the determination of themakeup of the protein. A global average is achieved, whether for whole-cellprotein extracts or for purified proteins.1. Lyophilize protein samples (e.g., half of the eluant from the Centri-Sep columnsin Subheading 3.2.3., or several micrograms of purified protein from Subheading3.2.2.) to dryness.2. Resuspend pellets in 1 mL of 5.4 M HCl with 10% thioglycolic acid to preservetryptophan during hydrolysis.3. Perform hydrolysis overnight, under a vacuum, at 110°C.4. Lyophilize the hydrolysates again, and resuspend in 50 µL of water.Hydrolysates can be analyzed by HPLC-ESI. In this case, the specific massof the natural and unnatural amino acid can be detected and followed as theyelute from the HPLC column. The relative ratios of the eluted masses can bedetermined as areas under the curves, and represent the relative amount of naturaland unnatural amino acids. The actual molar amounts of amino acids canalso be determined by generating a standard curve.3.3.1.2. HPLC–HPLC ANALYSIS OF HYDROLYZED PROTEIN SAMPLESAn alternative approach is to run serial HPLC samples.1. Inject hydrolysates onto a C-18 column and elute with the following program:a. 94% Buffer A and 6% Buffer B for 20 min.b. Switch by gradient to 98% Buffer A and 2% Buffer B during 10 min.c. Elute for an additional 15 min.d. Re-equilibrate the column to 94% Buffer A and 6% Buffer B in the last 5 minof the program.
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30 Bacher and EllingtonTable 1Incorporation of 6fW Into <strong>Protein</strong>Method of analysis Subheading Incorporation (%)Whole-cell protein extractHPLC-ESI 3.3.1.1 56.5HPLC-HPLC 3.3.1.2 66.7Purified GFPuvHPLC-ESI 3.3.1.1 68.6Average incorporation 64.0From ref. 4.3.3.1.1. HYDROLYSIS AND HPLC-ESI ANALYSIS OF PROTEINS CONTAININGUNNATURAL AMINO ACIDSAcid hydrolysis of protein samples allows for the determination of themakeup of the protein. A global average is achieved, whether for whole-cellprotein extracts or for purified proteins.1. Lyophilize protein samples (e.g., half of the eluant from the Centri-Sep columnsin Subheading 3.2.3., or several micrograms of purified protein from Subheading3.2.2.) to dryness.2. Resuspend pellets in 1 mL of 5.4 M HCl with 10% thioglycolic acid to preservetryptophan during hydrolysis.3. Perform hydrolysis overnight, under a vacuum, at 110°C.4. Lyophilize the hydrolysates again, and resuspend in 50 µL of water.Hydrolysates can be analyzed by HPLC-ESI. In this case, the specific massof the natural and unnatural amino acid can be detected and followed as theyelute from the HPLC column. The relative ratios of the eluted masses can bedetermined as areas under the curves, and represent the relative amount of naturaland unnatural amino acids. The actual molar amounts of amino acids canalso be determined by generating a standard curve.3.3.1.2. HPLC–HPLC ANALYSIS OF HYDROLYZED PROTEIN SAMPLESAn alternative approach is to run serial HPLC samples.1. Inject hydrolysates onto a C-18 column and elute with the following program:a. 94% Buffer A and 6% Buffer B for 20 min.b. Switch by gradient to 98% Buffer A and 2% Buffer B during 10 min.c. Elute for an additional 15 min.d. Re-equilibrate the column to 94% Buffer A and 6% Buffer B in the last 5 minof the program.
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