You have isolated a mixture of GST isoenzymes from rat liver tissue. You know th

Question

You have isolated a mixture of GST isoenzymes from rat liver tissue. You know that the mixture contains the following GST classes: GST alpha 2 GST alpha 3 GST omega 1 GST kappa 1 You have a gel filtration column with an exclusion limit of 2000 kD and an optimal separation range of 1 to 300 kD equilibrated with an appropriate physiological buffer. You also have both CM cellulose and DEAE cellulose ion exchange columns available for use with appropriate buffers for loading the sample and for a gradient elution like we used in the lab experiment. For ion exchange you have buffers available to use between pH 5 and 9, and you also have these same buffers at low salt and high salt concentrations. Your job is to try to separate these 4 from each other using different types of chromatography. Can you separate all isoforms from each other? You will need to search for the amino acid sequence, molecular weight and pls of each isoforms using the website, http://www.ncbi.nlm.nih.gov and the website, http://web.expasv.org/compute pi/. Print this information out as part of your answer. Then tell me whether you think gel Filtration with the given column will separate the isoforms Justify your answer. Next, tell me if and how you can use the different IEC columns with buffers at certain pHs (you need to pick buffers with certain pHs and tell me this in your answer) to separate the You need to pair up a buffer at a particular pH with a particular IEC column, either CM or DEAE cellulose. You may need to use several column runs in order to achieve separations and these need to be described. Some isoforms may not be able to be separated from each other. Tell me if that is the case. Draw the elution profiles when using the different columns. Include what isoforms would be in the void volume and the order of elution of those proteins that had originally bound to the column.

Explanation / Answer

GSTA2 glutathione S-transferase alpha 2

222 amino acid

1 MAEKPKLHYS NIRGRMESIR WLLAAAGVEF EEKFIKSAED LDKLRNDGYL MFQQVPMVEI

61 DGMKLVQTRA ILNYIASKYN LYGKDIKEKA LIDMYIEGIA DLGEMILLLP FTQPEEQDAK

121 LALIQEKTKN RYFPAFEKVL KSHGQDYLVG NKLSRADIHL VELLYYVEEL DSSLISSFPL

181 LKALKTRISN LPTVKKFLQP GSPRKPPMDE KSLEESRKIF RF

Using expasy PI = 8.51

Average Molecular weight of an amino acid is 110 dalton

So total MW =110x 222 =24420 dalton = 24.420 Kd .]

Using expasy Molecular weight = 25677.98 dalton =25.678 Kd

GST alpha 3

1 MKLFYKPGAC SLASHITLRE SGKDFTLVSV DLMKKRLENG DDYFAVNPKG QVPALLLDDG

61 TLLTEGVAIM QYLADSVPDR QLLAPVNSIS RYKTIEWLNY IATELHKGFT PLFRPDTPEE

121 YKPTVRAQLE KKLQYVNEAL KDEHWICGQR FTIADAYLFT VLRWAYAVKL NLEGLEHIAA

181 FMQRMAERPE VQDALSAEGL K

Using expasy Molecular weight = 22868.37dalton =22.868Kd

PI = 5.85

Glutathione Transferase Omega 1

240

1 MSGESARSLG KGSAPPGPVP EGSIRIYSMR FCPFAERTRL VLKAKGIRHE VININLKNKP

61 EWFFKKNPFG LVPVLENSQG QLIYESAITC EYLDEAYPGK KLLPDDPYEK ACQKMILELF

121 SKVPSLVGSF IRSQNKEDYA GLKEEFRKEF TKLEVLTNKK TTFFGGNSIS MIDYLIWPWF

181 ERLEAMKLNE CVDHTPKLKL WMAAMKEDPT VSALLTSEKD WQGFLELYLQ NSPEACDYGL

Using expasy Molecular weight = 27436.74 dalton =27.436 Kd

PI= 6.76

Glutathione S-transferase kappa 1

226

        1 MGPLPRTVEL FYDVLSPYSW LGFEILCRYQ NIWNINLQLR PSLITGIMKD SGNKPPGLLP

       61 RKGLYMANDL KLLRHHLQIP IHFPKDFLSV MLEKGSLSAM RFLTAVNLEH PEMLEKASRE

      121 LWMRVWSRNE DITEPQSILA AAEKAGMSAE QAQGLLEKIA TPKVKNQLKE TTEAACRYGA

      181 FGLPITVAHV DGQTHMLFGS DRMELLAHLL GEKWMGPIPP AVNARL

Using expasy Molecular weight = 25496.86 dalton =25.496 Kd

PI =8.91

So we can purify the all protein by using the provided ion exchange chromatography.

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