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These are the user uploaded subtitles that are being translated: 0 00:00:00,380 --> 00:00:00,820 NATHANIEL: Hi. 1 00:00:00,820 --> 00:00:02,220 My name's Nathaniel. 2 00:00:02,220 --> 00:00:05,460 As a scientist at MIT, I study how ultraviolet light damages the proteins 3 00:00:05,460 --> 00:00:06,750 in the human lens. 4 00:00:06,750 --> 00:00:10,210 And for my experiments, I purify human lens proteins from bacterial E. coli 5 00:00:10,210 --> 00:00:12,860 cells designed specifically to express human proteins. 6 00:00:12,860 --> 00:00:16,090 In this demonstration video, I'm going to show you how biochemists use column 7 00:00:16,090 --> 00:00:18,840 chromatography to purify a variety of proteins. 8 00:00:18,840 --> 00:00:23,030 In specific, today we'll be purifying GFP, green fluorescent protein. 9 00:00:23,030 --> 00:00:25,810 In lecture, Professor Landers talked about the incredible diversity of 10 00:00:25,810 --> 00:00:28,030 structure and functioning that proteins have. 11 00:00:28,030 --> 00:00:30,220 Protein purification is a critical step to understand that 12 00:00:30,220 --> 00:00:31,600 structure and function. 13 00:00:31,600 --> 00:00:34,300 By the end of this video, you'll be able to describe how biochemists use 14 00:00:34,300 --> 00:00:37,230 column chromatography to purify any number of proteins. 15 00:00:37,230 --> 00:00:39,940 Before we get started, let's talk about we'll be doing today. 16 00:00:39,940 --> 00:00:42,710 How do we purify one specific protein from all the other 17 00:00:42,710 --> 00:00:43,710 proteins in the cell. 18 00:00:43,710 --> 00:00:46,700 As Professor Landers discussed, scientists to start by breaking open, 19 00:00:46,700 --> 00:00:49,110 or lysing cells to release all the proteins. 20 00:00:49,110 --> 00:00:51,590 Once they have this mixture of proteins, how do they isolate one 21 00:00:51,590 --> 00:00:53,290 specific protein from all the others? 22 00:00:53,290 --> 00:00:56,300 Scientists can use a technique called column chromatography to take 23 00:00:56,300 --> 00:00:59,080 advantage of the different properties of their specific protein of interest, 24 00:00:59,080 --> 00:01:00,540 like size and charge. 25 00:01:00,540 --> 00:01:02,160 Some proteins are larger than others. 26 00:01:02,160 --> 00:01:04,830 Some proteins are more hydrophilic or hydrophobic than others. 27 00:01:04,830 --> 00:01:07,670 We can separate proteins by flowing a protein mixture through a matrix of 28 00:01:07,670 --> 00:01:08,720 beads in a column. 29 00:01:08,720 --> 00:01:11,260 The scientists select the beads for the column and the binding buffer 30 00:01:11,260 --> 00:01:13,880 based on the properties of the protein of interest. 31 00:01:13,880 --> 00:01:16,400 The protein of interest binds to the beads based on its affinity for the 32 00:01:16,400 --> 00:01:17,880 beads in the column. 33 00:01:17,880 --> 00:01:20,670 To remove other unwanted bound proteins that are more weakly 34 00:01:20,670 --> 00:01:23,810 associated with the column, scientists can wash the column with a different 35 00:01:23,810 --> 00:01:26,650 buffer that pushes the weakly bound proteins off. 36 00:01:26,650 --> 00:01:29,440 When removing the protein from the column, we use a third buffer, and 37 00:01:29,440 --> 00:01:32,020 collect fractions of liquid coming off the column. 38 00:01:32,020 --> 00:01:34,600 For most proteins, scientists don't know which fraction contains the 39 00:01:34,600 --> 00:01:35,590 protein of interest. 40 00:01:35,590 --> 00:01:38,300 They have to test each fraction for a specific activity. 41 00:01:38,300 --> 00:01:41,560 For today, we'll take advantage of the unique fluorescent property of GFP, 42 00:01:41,560 --> 00:01:43,820 and use its green glow to track the protein throughout our 43 00:01:43,820 --> 00:01:45,470 purification steps. 44 00:01:45,470 --> 00:01:47,900 Do you know that GFP originally came from jellyfish? 45 00:01:47,900 --> 00:01:50,950 GFP is a really cool protein because it fluoresces green and serves as a 46 00:01:50,950 --> 00:01:52,660 valuable tool for scientists. 47 00:01:52,660 --> 00:01:55,850 Scientists have found that the protein has a beta barrel structure, a barrel 48 00:01:55,850 --> 00:01:59,070 shape made up of beta strands that you heard about from Professor Landers. 49 00:01:59,070 --> 00:02:03,490 Scientists copy the gene encoding GFP and place into the bacteria E. coli. 50 00:02:03,490 --> 00:02:06,180 So where do we produce this GFP for purification? 51 00:02:06,180 --> 00:02:08,690 We need to make many copies of the protein to efficiently isolate the 52 00:02:08,690 --> 00:02:09,880 protein of interest. 53 00:02:09,880 --> 00:02:10,800 Let's get started. 54 00:02:10,800 --> 00:02:14,550 I have E. coli cells that are designed to express GFP. 55 00:02:14,550 --> 00:02:15,800 They grew and divided overnight. 56 00:02:24,380 --> 00:02:26,310 I also have cells that don't express GFP. 57 00:02:26,310 --> 00:02:28,830 We'll collect these cells too as a comparison. 58 00:02:28,830 --> 00:02:32,300 Because it's not ideal to isolate GFP from jellyfish in the lab, we can make 59 00:02:32,300 --> 00:02:36,470 many copies of GFP by expressing the protein in the bacterium E. coli. 60 00:02:36,470 --> 00:02:39,430 How do we get bacteria to make protein from a jellyfish? 61 00:02:39,430 --> 00:02:43,610 Scientists copy the gene encoding GFP and place it in the bacterium E. coli. 62 00:02:43,610 --> 00:02:46,300 You'll learn more about this process of cloning later in the course, as 63 00:02:46,300 --> 00:02:49,460 well as how scientists use GFP in research. 64 00:02:49,460 --> 00:02:52,860 We spin the cells at a high speed in these tubes, so that the cells go to 65 00:02:52,860 --> 00:02:54,570 the bottom as a pellet. 66 00:02:54,570 --> 00:02:58,380 How do we know that our bacterial cells really are expressing GFP? 67 00:02:58,380 --> 00:03:01,950 We can use this really cool property of GFP that it fluoresces. 68 00:03:01,950 --> 00:03:03,800 I have this UV lamp here. 69 00:03:03,800 --> 00:03:06,990 And when we shine the UV light on the cells that are expressing GFP, they 70 00:03:06,990 --> 00:03:07,890 should glow green. 71 00:03:07,890 --> 00:03:09,190 Let's test our cells. 72 00:03:09,190 --> 00:03:12,050 I'm removing the liquid from the tube and saving the cell pellets. 73 00:03:12,050 --> 00:03:15,730 Let's shine the light on the cells that are not supposed to express GFP. 74 00:03:15,730 --> 00:03:17,210 What do you expect to see? 75 00:03:17,210 --> 00:03:18,000 Nothing, right? 76 00:03:18,000 --> 00:03:19,560 No green fluorescence. 77 00:03:19,560 --> 00:03:22,300 Let's shine the light on the cells that are supposed to express GFP. 78 00:03:22,300 --> 00:03:23,150 Check it out. 79 00:03:23,150 --> 00:03:24,270 Green glow. 80 00:03:24,270 --> 00:03:27,600 So how will we lyse our E. coli cells now to get at that mixture of proteins 81 00:03:27,600 --> 00:03:28,955 within, including GFP? 82 00:03:31,670 --> 00:03:34,410 I'm re-suspending the cell pellets in a buffer by pipetting up and down 83 00:03:34,410 --> 00:03:36,110 repeatedly. 84 00:03:36,110 --> 00:03:38,920 Now I'm adding an enzyme that breaks down cell walls called lysozyme. 85 00:03:42,470 --> 00:03:44,530 Next, I'll freeze then thaw the cells quickly. 86 00:03:49,660 --> 00:03:52,340 This lyses the cells, releasing all the proteins, including 87 00:03:52,340 --> 00:03:54,360 GFP, from the cells. 88 00:03:54,360 --> 00:03:57,450 Afterward, I'll centrifuge the samples again to separate insoluble cell 89 00:03:57,450 --> 00:03:58,700 debris from the soluble proteins. 90 00:04:02,960 --> 00:04:05,960 So how will we separate GFP from all the other soluble proteins in the 91 00:04:05,960 --> 00:04:07,610 bacterial cell? 92 00:04:07,610 --> 00:04:10,260 Now that I have the cell debris separated from the soluble proteins, 93 00:04:10,260 --> 00:04:13,560 I'll use a small column packed with tiny beads to separate the proteins by 94 00:04:13,560 --> 00:04:15,190 a specific property. 95 00:04:15,190 --> 00:04:17,290 In this case, I'm using an affinity column that 96 00:04:17,290 --> 00:04:19,089 contains hydrophobic beads. 97 00:04:19,089 --> 00:04:21,810 GFP contains many hydrophobic amino acids. 98 00:04:21,810 --> 00:04:25,600 In a high-salt buffer, the hydrophobic parts of GFP move to the exterior of 99 00:04:25,600 --> 00:04:28,010 the protein, and GFP binds to the beads as the 100 00:04:28,010 --> 00:04:29,720 protein enters the column. 101 00:04:29,720 --> 00:04:33,110 The other bacterial proteins with fewer hydrophobic amino acids flow 102 00:04:33,110 --> 00:04:35,870 past the beads and through the column. 103 00:04:35,870 --> 00:04:38,190 Let's prepare our column for purification. 104 00:04:38,190 --> 00:04:41,310 I'm adding a buffer with the same salt concentration as our protein solution 105 00:04:41,310 --> 00:04:43,200 to equilibrate the beads. 106 00:04:43,200 --> 00:04:45,430 I'm removing the cap at the bottom of the column to allow the 107 00:04:45,430 --> 00:04:46,680 buffer to flow through. 108 00:04:50,070 --> 00:04:53,540 With that done, I recap the bottom of the column. 109 00:04:53,540 --> 00:04:56,490 Now I'm adding my cell extract of soluble proteins and a high salt 110 00:04:56,490 --> 00:04:59,180 buffer to the top of the column. 111 00:04:59,180 --> 00:05:02,400 Using this UV lamp, I should be able to detect any GFP. 112 00:05:02,400 --> 00:05:03,420 Take a peek-- 113 00:05:03,420 --> 00:05:05,160 glowing green! 114 00:05:05,160 --> 00:05:07,450 So we can see a ring of green fluorescence at the top of the column 115 00:05:07,450 --> 00:05:10,360 showing that GFP has bound to the top of the column. 116 00:05:10,360 --> 00:05:12,860 So how will we make sure that all the other bacterial proteins are removed 117 00:05:12,860 --> 00:05:13,920 from the column? 118 00:05:13,920 --> 00:05:17,550 I'm adding a wash buffer with a medium salt concentration to our column. 119 00:05:17,550 --> 00:05:20,990 Any proteins with less hydrophobic exteriors than GFP did not bind as 120 00:05:20,990 --> 00:05:23,300 tightly to the hydrophobic beads, and will flow out of the 121 00:05:23,300 --> 00:05:25,030 column during washing. 122 00:05:25,030 --> 00:05:28,230 The hydrophobic exterior of GFP has a higher affinity for the beads and 123 00:05:28,230 --> 00:05:29,205 remains bound. 124 00:05:29,205 --> 00:05:32,820 So now that all bacterial proteins are washed away, how do we remove GFP from 125 00:05:32,820 --> 00:05:34,250 the beads on the column? 126 00:05:34,250 --> 00:05:37,190 Remember, the GFP bound to beads in the column because the beads in the 127 00:05:37,190 --> 00:05:40,300 GFP exterior were currently both hydrophobic. 128 00:05:40,300 --> 00:05:43,020 I can remove GFP from the beads by adding a buffer with low salt 129 00:05:43,020 --> 00:05:43,990 concentration. 130 00:05:43,990 --> 00:05:48,000 So what do you think the low salt concentration solution will do to GFP? 131 00:05:48,000 --> 00:05:51,310 The low salt concentration will bring the hydrophilic residues of GFP to the 132 00:05:51,310 --> 00:05:53,900 surface, so the protein will no longer bind to hydrophobic 133 00:05:53,900 --> 00:05:54,970 beads in the column. 134 00:05:54,970 --> 00:05:58,180 I'm collecting fractions of what comes off the column in several tubes. 135 00:05:58,180 --> 00:06:02,140 Let's see if the GFP is moving through the column now with our UV light. 136 00:06:02,140 --> 00:06:06,080 Yes, I can see the GFP moving through the column. 137 00:06:06,080 --> 00:06:08,480 Which tube contains the GFP protein? 138 00:06:08,480 --> 00:06:11,750 I do not know by only looking at the liquid collected off the column. 139 00:06:11,750 --> 00:06:14,290 For most proteins, we would have to test each fraction with an activity 140 00:06:14,290 --> 00:06:16,880 assay or another protein detection assay. 141 00:06:16,880 --> 00:06:18,610 But remember, we're purifying a protein with the 142 00:06:18,610 --> 00:06:20,440 unique ability to fluoresce. 143 00:06:20,440 --> 00:06:22,990 Let's check which fraction I collected contains the GFP by using 144 00:06:22,990 --> 00:06:24,010 the UV light again. 145 00:06:24,010 --> 00:06:26,150 I can see that most of the green fluorescence is in tube four. 146 00:06:26,150 --> 00:06:28,650 So GFP eluted in fraction four. 147 00:06:28,650 --> 00:06:31,880 I've now purified GFP for most of the other bacterial proteins. 148 00:06:31,880 --> 00:06:33,540 And you've learned something about biochemistry and 149 00:06:33,540 --> 00:06:34,700 how we purify proteins. 150 00:06:34,700 --> 00:06:35,950 I hope you had fun. 12768

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