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Problem of recombinant bacterial membrane anchored protein


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#1 vels

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Posted 13 April 2011 - 07:19 AM

Hi all,

I just cloned a bacterial membrane enchored protein without N-terminal signal peptide in pET15b vector and expressed in BL21 host. The expected size of recombinant protein should be 80.3 kDa. After induction, I just boiled the cell pellet of a positive clone in sample buffer for 8 min, centrifuged for 10 min and loaded onto 12% SDS gel. This gel is just to confirm the expression of recombinant protein. But, instead of 80 kDa recombinant protein, I saw a big band at 37 kDa and another big band at 33 kDa. Then, I purified the expressed protein using metal affinity resin specific for His-tag. The purified protein was mixed with sample buffer, boiled for 8 min, centrifuged for 5 min and loaded onto 12% SDS gel. Then also, I saw 2 pure bands at 37 kDa and 33 kDa. Why, I am not getting intact 80 kDa band instead two bands at 37 kDa and 33 kDa?

#2 protolder

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Posted 13 April 2011 - 10:26 PM

Hi all,

I just cloned a bacterial membrane enchored protein without N-terminal signal peptide in pET15b vector and expressed in BL21 host. The expected size of recombinant protein should be 80.3 kDa. After induction, I just boiled the cell pellet of a positive clone in sample buffer for 8 min, centrifuged for 10 min and loaded onto 12% SDS gel. This gel is just to confirm the expression of recombinant protein. But, instead of 80 kDa recombinant protein, I saw a big band at 37 kDa and another big band at 33 kDa. Then, I purified the expressed protein using metal affinity resin specific for His-tag. The purified protein was mixed with sample buffer, boiled for 8 min, centrifuged for 5 min and loaded onto 12% SDS gel. Then also, I saw 2 pure bands at 37 kDa and 33 kDa. Why, I am not getting intact 80 kDa band instead two bands at 37 kDa and 33 kDa?

Hola it is extrange that both fragments were purified in metal affinity resin. Why dont you made a WB with anti-His to see if two bands have the tag, if only one has it check the DNA sequence in the broken area if there is any protease site or any termination codon. Buena suerte

#3 vels

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Posted 18 April 2011 - 10:44 AM

Thank you Veteran for your suggestion. I thought that may be boiling is cleaving the band. So, in my next 12% SDS gel, I loaded the purified His tag protein without boiling. Then, I got a single band at 35 kDa region. Now, there are no 2 bands. Please tell me the reason for the reduced molecular weight of my expressed protein? My expected size is 80 kDa, but after expression and purification, I got a pure band at 35 kDa only. What is the reason for this? In order to see whether there is any stop codon, I sent the plasmid for sequencing and I am waiting for the result.











Hi all,

I just cloned a bacterial membrane enchored protein without N-terminal signal peptide in pET15b vector and expressed in BL21 host. The expected size of recombinant protein should be 80.3 kDa. After induction, I just boiled the cell pellet of a positive clone in sample buffer for 8 min, centrifuged for 10 min and loaded onto 12% SDS gel. This gel is just to confirm the expression of recombinant protein. But, instead of 80 kDa recombinant protein, I saw a big band at 37 kDa and another big band at 33 kDa. Then, I purified the expressed protein using metal affinity resin specific for His-tag. The purified protein was mixed with sample buffer, boiled for 8 min, centrifuged for 5 min and loaded onto 12% SDS gel. Then also, I saw 2 pure bands at 37 kDa and 33 kDa. Why, I am not getting intact 80 kDa band instead two bands at 37 kDa and 33 kDa?

Hola it is extrange that both fragments were purified in metal affinity resin. Why dont you made a WB with anti-His to see if two bands have the tag, if only one has it check the DNA sequence in the broken area if there is any protease site or any termination codon. Buena suerte






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