MADELINE+J

__ Title: An Expressive, Pure, Character with Purple Protein __

__ Introduction: __ Protein production and purification technique has been essential to biological and biomedical research. Typically //E. coli// is used as the expression host for the initial recombinant production of protein, with BL21(DE3) strain ideal for high-level protein production. In order to select for bacterial uptake of plasmid, antibiotics such as ampicillin are added in conjunction to antibiotic-resistant plasmids. Affinity tags, such as commonly used N-terminal hexahistidine tags, greatly aid in protein purification as the oligohistidine-tagged proteins can be purified with little difficulty, and the histidine tag rarely alters the characteristics of the protein such as structure and solubility.[1] IMAC (immobilized metal affinity chromatography) is a chromatographic procedure used in conjunction to the hexahistidine tags and controlled concentrations of imidazole to isolate the protein of interest as well draw out waste debris from supernatent.[1,2] The purpose of this set of experiments (Protein Expression, Purification, Characterization) was to successfully express our protein gene gbr-22 in E. coli BL21(DE3) by starter culture and amplify in large culture, isolate our purple protein, and assess the quantity and quality of our yield. It was hypothesized that the majority of the gbr-22 protein would be isolated in Elution #1.

__ Materials & Methods: __

 Competent BL21(DE3) bacterial cells (NEB New England Biolabs) were transformed with pGEM-gbr22 plasmid via heat shock (42°C, 45sec) with ampicillin. A separate plate was used as a negative control with no DNA insert. Plates were grown in 37oC incubator overnight. An individual purple colony was transferred LB (5mL)/ ampicillin(10.1 m L) media and grown in starter culture. Seven hours later, protein was further expressed in large culture with .525 mL of starter culture, 25mL LB and 51.1 m L ampicillin. The large culture was grown for 16 hours at 37oC and 200-350 rpm.  Cells were then harvested after 16 hours by spinning down the contents of large culture at 10 minutes, 5000rpm, at 40C, and removing the liquid spent media to isolate cell pellet. E. coli cells were lysed and resuspended with 2.4 mL of 1x PBS and a final concentration of 1mg/mL for lysozyme. Cells were stored in -20o C. The 2.5 mL suspension was incubated 20 min at room temperature. 2 m l of Cyanase was then added, and lysate was further incubated for 15 minutes. Lysate was centrifuged for 20 minutes at 14,000 rpm at 4oC to clarify. Liquid supernatant was isolated from cell debris and filtered via .45 m m syringe filter (EMD Millipore, Billerica, MA).  Soluble fraction was isolated through Ni-NTA affinity purification with .5mL Ni-NTA resin/buffer mix. Flow waste, wash (5 ml of 20 mM imidazole in 1x PBS buffer) and Elutions(5 mL of 250mM imidazole buffer 1x PBS) 1 and 2 were collected from 20 ml Bio-Rad chromatography Econo column. Nanodrop spectrophotometer(Thermo Scientific, Wilmington, DE) used to analyze absorbance of Elution 1.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted on samples with 1xTGS buffer, 1x loading buffer for 25 minutes. Partner's samples 1-6 (S1-S6) were tested along with personal S4 and S6. Molecular weight standard ( Page-Ruler, #26616,Thermo Scientific) used in order to estimate protein size. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Gel was washed, stained with Imperial protein stain, washed overnight, and dried 75 ° <span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5; text-align: justify;">C on Gradient cycle for 1.5 hours.

This is a good summary of the process, but also remember to explain why the steps were taken. Try to make it flow better and use transitions. this may seem like English class stuff but it really does help strengthen the paper as a whole. A little analysis here and there will also help this seem less choppy.

__ Results: __

Figure 1: Agar plate containing ampicilin incubated at 37 degrees Celsius overnight of originally E.coli BL21 (DE3) with pGEM-gbr22 plasmid insert smear.

Figure 2: Agar plate containing ampicilin incubated at 37 degrees Celsius overnight containing E. coli BL21(DE3) with no plasmid insert.

Figure 3: Agar plate containing ampicilin incubated at 37 degrees Celsius overnight of originally E.coli BL21 (DE3) with pGEM-gbr22 plasmid insert smear. Plate credited to Kavya K. and Muhammed A. Actual plate that was used.

<span style="background-color: white; font-family: Arial; font-size: 10pt; line-height: 0px; overflow: hidden;">
 * Figure 4: E.coli B21(DE3) with pGEM-gbr22 plasmid insert <span style="background-color: white; font-family: Arial; font-size: 10pt; line-height: 0px; overflow: hidden;">large culture grown in LB media supplemented with 100 micrograms/mL ampicillin for 16-24 hours in a shaking incubator at 37 degrees Celsius and 200-350 rpm.

<span style="background-color: white; font-family: Arial; font-size: 10pt; line-height: 0px; overflow: hidden;">

Figure 5: Purple pellet containing harvested E.coli B21(DE3) with pGEM-gbr22 plasmid insert after centrifuged at 4 degrees Celsius for 10 minutes at 5000 rpm. Pellet shown credited to D'Ondria Peters.







**<span style="font-family: Arial,sans-serif; font-size: 11pt;">Beer’s law calculations. A=Ebc **

**<span style="font-family: Arial,sans-serif; font-size: 11pt;">Concentration of protein using 280nm **
 * **<span style="font-family: Arial,sans-serif; font-size: 11pt;">At wavelength =280nm, b=1cm, E=28850 L m-1cm-1, A=averaged absorbance readings=355 a.u., Molecular Weight=25794.2g/mol **

**<span style="font-family: Arial,sans-serif; font-size: 11pt;">Using Maximal wavelength at 574nm **
 * **<span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5; text-align: justify;">concentration=(A/Eb)*MW=.236g/L=.236mg/mL **
 * **<span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5; text-align: justify;">yield=c*V=.235*4.7mL=1.1092g **
 * **<span style="font-family: Arial,sans-serif; font-size: 11pt;">At wavelength =574nm, b=.1 cm, E=118300 L m-1cm-1, A=averaged absorbance readings of .071 au and .077 au=.074 a.u., Molecular Weight=25794.2g/mol **


 * **<span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5; text-align: justify;">concentration=(A/Eb)*MW=.161 g/L =.161 mg/mL **
 * **<span style="font-family: Arial,sans-serif; font-size: 11pt; line-height: 1.5; text-align: justify;">yield=c*V=.161*4.7mL=.758 g **

__ Discussion: __

<span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Bacteria colonization was not successful as seen in Figure 1. Experiment proceeded with sample obtained from another plate (Figure 3). Pellet size from expression weighed .77g, which was satisfactory. Gel produced was destained cleanly. Purity of sample was satisfactory at about 75% in Figure 8. Contamination was evident by presence of a second band; however, the second band’s lack of intensity indicated contamination was limited. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Lysozyme’s function in digesting bacterial cell walls allowed for release of purple protein into solution. Benzonase/Cyanase was used to digest the DNA/RNA, allowing for the removal of nucleic acid from lysate. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">HIS tag system was used to isolate protein from cell debris/other contaminants through controlled binding affinity. Nickel from the Ni-NTA matrix could interact with the 6xHis tag as well as imidazole. At low concentrations of imidazole, the majority of purple protein was bound to the Ni-NTA matrix and thus caught by the filter, separating smaller contaminants into flow waste. At high concentrations of imidazole, the majority of protein was released into elutions while imidazole outcompeted for binding. Thus wash and elution buffers differed only in imidazole concentrations. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">S1 contained the contents of lysed E. coli cells expressing gbr22. S2 contained soluble contents of the bacteria after centrifugation of cell debris pellet. S3 contained affinity chromatography flow. S4 contained flow after wash buffer (20 mM imidazole in 1x PBS buffer) was loaded. S5 and S6 were elutions with buffer of 250 mM imidazole. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Molecular weight of protein from the gel was estimated to be about 26,000g/mol essentially equal to known molecular weight of 25,794.2g/mol as the low precision nature in reading protein ladder. <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Major sources of error for the experimental procedure include loss of .5mL in Elution 2, contamination of samples as evident in Elution 1 in Figure 8, and leftover protein in column filter potentially resulting in lower yield. Contamination could have resulted from improper aseptic technique as well as failure in perfect Ni-NTA affinity purification (Explain) <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">. Calculation of concentration was subject to low significant figures of readings. ( again good analysis but this is very choppy and does not transition from topic to topic well) __ Conclusions: __ <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">Aside from initial efforts to grow the starter culture (Figure 1), protein gbr22 was successfully expressed, purified and characterized in the past three labs as demonstrated in Figure 8. Future directions would involve further purification of Elution 1 and ultimately cultivation (completion is a better word here) <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">of an enzyme essay (better get writing then (it's ASSAY). <span style="font-family: Arial,sans-serif; font-size: 11pt; text-align: justify;">In VDS, eventual directions seek use of these techniques in order to supply and study disease-relevant proteins. Future directions are anticipated to come across more difficulties, such as issues with protein folding, transformation of bacteria, and prevention of contaminants. (specifically how will this lab be useful to your research in the future?) __ References: __

[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards,A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.;Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.;Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.;Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.;Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46. [2] Bornhorst, B. J.; Falke, J. J., Reprint of: Purification of Proteins Using Polyhistidine Affinity Tags. // Protein Expr Purif //** 2011 **.