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3. A student in my lab was trying to express the cytosolic (not membrane bound) enzyme PseG and decided to express the enzyme as a His fusion (PseG-His) to ease purification. In his initial experiments, he (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a gentle lysis buffer (one that would solubilize folded proteins), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS-PAGE analysis. In his first expression experiment, he collected the data shown in Figure 1: kD 58 46 kD MW WC PP SS 175 80 30- 25 10- Coomassie-stained protein gel a. From these initial data, what can you conclude about the following features of PseG-His6 in E. coli? Expressed? Soluble? Folded? 10 In a follow-up experiment, my student (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a harsh lysis buffer that included 6 M urea (a detergent), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS- PAGE analysis. In this second experiment, he collected the data shown in Figure 2: ← PseG-His6 fusion WC PS expression was Figure 1. Protein experiment #1. PseG-His6 expressed in E. coli. E. coli were then burst open using a gentle lysis buffer that would solubilize folded proteins. The presence of PseG-Hise in whole- cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis. Pellet and supernatant fractions were run in duplicate. ← PseG-His6 fusion Figure 2. Protein expression experiment #2. PseG-His6 was expressed in E. coli. E. coli were then burst open using a harsh lysis buffer that included 6 M urea (a detergent). The presence of PseG-Hise in whole-cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis.

3. A student in my lab was trying to express the cytosolic (not membrane bound) enzyme PseG and decided to express the enzyme as a His fusion (PseG-His) to ease purification. In his initial experiments, he (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a gentle lysis buffer (one that would solubilize folded proteins), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS-PAGE analysis. In his first expression experiment, he collected the data shown in Figure 1: kD 58 46 kD MW WC PP SS 175 80 30- 25 10- Coomassie-stained protein gel a. From these initial data, what can you conclude about the following features of PseG-His6 in E. coli? Expressed? Soluble? Folded? 10 In a follow-up experiment, my student (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a harsh lysis buffer that included 6 M urea (a detergent), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS- PAGE analysis. In this second experiment, he collected the data shown in Figure 2: ← PseG-His6 fusion WC PS expression was Figure 1. Protein experiment #1. PseG-His6 expressed in E. coli. E. coli were then burst open using a gentle lysis buffer that would solubilize folded proteins. The presence of PseG-Hise in whole- cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis. Pellet and supernatant fractions were run in duplicate. ← PseG-His6 fusion Figure 2. Protein expression experiment #2. PseG-His6 was expressed in E. coli. E. coli were then burst open using a harsh lysis buffer that included 6 M urea (a detergent). The presence of PseG-Hise in whole-cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis.

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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3. A student in my lab was trying to express the cytosolic (not membrane bound) enzyme PseG and decided to express the enzyme as a Hisí fusion (PseG-His) to ease purification. In his initial experiments, he (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a gentle lysis buffer (one that would solubilize folded proteins), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS-PAGE analysis. In his first expression experiment, he collected the data shown in Figure 1: kD 58 46 kD MW WC PP 175 80 58 46 30 25 │I T||| || | 10- Coomassie-stained protein gel a. From these initial data, what can you conclude about the following features of PseG-His6 in E. coli? Expressed? Soluble? Folded? 10->> In a follow-up experiment, my student (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a harsh lysis buffer that included 6 M urea (a detergent), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS- PAGE analysis. In this second experiment, he collected the data shown in Figure 2: SS WC PS ← PseG-His6 fusion Coomassie-stained protein gel expression was Figure 1. Protein experiment #1. PseG-His6 expressed in E. coli. E. coli were then burst open using a gentle lysis buffer that would solubilize folded proteins. The presence of PseG-His6 in whole- cells (WC) before lysis, and pellet (P) and supernatant (S) fractions_post-lysis, was assessed by SDS-PAGE analysis. Pellet and supernatant fractions were run in duplicate. PseG-His6 fusion Figure 2. Protein expression experiment #2. PseG-His6 was expressed in E. coli. E. coli were then burst open using a harsh lysis buffer_that included 6 M urea (a detergent). The presence of PseG-His in whole-cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis.
Transcribed Image Text:3. A student in my lab was trying to express the cytosolic (not membrane bound) enzyme PseG and decided to express the enzyme as a Hisí fusion (PseG-His) to ease purification. In his initial experiments, he (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a gentle lysis buffer (one that would solubilize folded proteins), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS-PAGE analysis. In his first expression experiment, he collected the data shown in Figure 1: kD 58 46 kD MW WC PP 175 80 58 46 30 25 │I T||| || | 10- Coomassie-stained protein gel a. From these initial data, what can you conclude about the following features of PseG-His6 in E. coli? Expressed? Soluble? Folded? 10->> In a follow-up experiment, my student (1) expressed the protein in bacterial cells (E. coli), (2) lysed the bacteria using a harsh lysis buffer that included 6 M urea (a detergent), and (3) assessed the protein's presence in whole-cells (WC) pre-lysis, and pellet (P) versus soluble (S) cell fractions post-lysis, by SDS- PAGE analysis. In this second experiment, he collected the data shown in Figure 2: SS WC PS ← PseG-His6 fusion Coomassie-stained protein gel expression was Figure 1. Protein experiment #1. PseG-His6 expressed in E. coli. E. coli were then burst open using a gentle lysis buffer that would solubilize folded proteins. The presence of PseG-His6 in whole- cells (WC) before lysis, and pellet (P) and supernatant (S) fractions_post-lysis, was assessed by SDS-PAGE analysis. Pellet and supernatant fractions were run in duplicate. PseG-His6 fusion Figure 2. Protein expression experiment #2. PseG-His6 was expressed in E. coli. E. coli were then burst open using a harsh lysis buffer_that included 6 M urea (a detergent). The presence of PseG-His in whole-cells (WC) before lysis, and pellet (P) and supernatant (S) fractions post-lysis, was assessed by SDS-PAGE analysis.
b. What can you conclude about the following features of PseG-His。 in E. coli in this follow-up experiment? Next, my student set out to purify the PseG-His present in the supernatant from the harsh lysis (obtained in Figure 2). He loaded the lysate onto a Ni+² column, rinsed the column with buffer containing increasing concentrations of imidazole, and analyzed fractions for the presence of PseG-His6: C. kD 30 25 || Expressed? Soluble? Folded? 10-> [imidizole] 50-100 mM 200 mM imidizole PseG-His fusion Figure 3. Fractions from a Ni+²-column analyzed by SDS-PAGE. The column was loaded with E. coli lysate and subsequently rinsed with buffer containing increasing concentrations of imidazole as indicated. imidazole Coomassie-stained protein gel What type of purification is this? What happened when 200 mM imidazole was added to the column? Why? d. Is the purified protein obtained in Figure 3 active? If not, what could my student do to help this protein gain activity?
Transcribed Image Text:b. What can you conclude about the following features of PseG-His。 in E. coli in this follow-up experiment? Next, my student set out to purify the PseG-His present in the supernatant from the harsh lysis (obtained in Figure 2). He loaded the lysate onto a Ni+² column, rinsed the column with buffer containing increasing concentrations of imidazole, and analyzed fractions for the presence of PseG-His6: C. kD 30 25 || Expressed? Soluble? Folded? 10-> [imidizole] 50-100 mM 200 mM imidizole PseG-His fusion Figure 3. Fractions from a Ni+²-column analyzed by SDS-PAGE. The column was loaded with E. coli lysate and subsequently rinsed with buffer containing increasing concentrations of imidazole as indicated. imidazole Coomassie-stained protein gel What type of purification is this? What happened when 200 mM imidazole was added to the column? Why? d. Is the purified protein obtained in Figure 3 active? If not, what could my student do to help this protein gain activity?
Expert Solution
Step 1

The expression of proteins in a prokaryotic or eukaryotic cell involves transcription and translation progress of the gene of interest n the cell in which the protein is being expressed.

The bacterial expression system can act as rapid and simple systems of expressing large amounts of recombinant eukaryotic proteins. 

However some recombinant proteins however tend to form inclusion bodies upon their expression in bacterial cells. 

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