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Expression Purification and Characterization of KsgA Protein
Deleah Pettie
Lab Partner : Michael Polzin
Lab Section A
111!"#1$
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Abstract
%his stud& 'as perfor(ed in order to sho'
ho' a desired protein of interest can be
obtained b& starting 'ith the gene of
interest then using techni)ues of genetic
(odification and cloning in order to a(plif&
the desired gene then express the protein
fro( the ne' plas(id and purif& the sa(ple
in order to obtain the desired product* %he
ob+ecti,e of the stud& 'as to see if the
cloning expression and purifications
techni)ues 'ould be useful in obtaining the
KsgA protein fro( a ,ector* %he results
fro( the stud& sho' the KsgA protein 'as
present in the PC- sa(ples so thea(plification of the gene 'as successfull&
sho'n b& the presence of a bright band in
the agarose gel* %he o,er expression of the
KsgA protein 'as seen in the ti(e induction
gel sa(ples the band for each ti(e induction
beca(e dar.er sho'ing a higher presence of
the protein* KsgA 'as detected in (an&
sa(ples of the L&sate of cells and in t'o
fractions of the protein purification process
this 'as confir(ed 'ith an i((unoblotcolori(etric blot and SDS/PA0E to sho'
the purit& of the product* ased on the
results the ,arious isolation2 expression2 and
purification techni)ues 'ere successful in
producing the KsgA protein*
Introduction
Man& ad,ances ha,e led to the de,elop(ent
of techni)ues that can be used to express
proteins at higher le,els and i(pro,ing &ield
also other techni)ues ha,e been de,eloped
in order to geneticall& (odif& proteins so
the purification process is si(pler* %hese
procedures are de(onstrated in this stud& of
protein expression purification and
characterization the protein of interest KsgA
has been (odified 'ith 3/4is tags in order
to help the purification process 'ith Metal
Chelate Affinit& Chro(atograph& 5ic.el is
used in the colu(n 'hich causes an
interaction 'ith the 4is/tag allo'ing for
better purification of the protein* %he 4is/
tags are also useful in helping detect the
protein of interest through the use of
antibodies* 6P%0 is used in order to help the
o,erexpression of the KsgA protein through
ti(e induction* All of these procedures are
perfor(ed in order to see ho' efficient it is
to geneticall& (odif& genes in order to
obtain desired product fro( cloning and
purification*
Materials and MethodColony PCR
7i,e pol&(erase Chain -eaction
a(plification reactions 'ere prepared for
colonies plated b& the teaching assistants
each plate labeled A22C and D and the fifth
reaction has no colon& and is the negati,e
control* 7or the fi,e reactions a (aster (ix
'as prepared 1"8ul total for the fi,e
reactions containing 1"*8 ul of PC- 1#9
uffer *8 ul of MgCl""8(M stoc.concentration2 8ul d5%Ps 8(M stoc.2 8ul
for pri(ers for'ard and re,erse2 ;"*8 ul of
sterile 'ater and 1*8 ul of %a) added to the
fi,e reactions indi,iduall& e)ualing *8 ul
total* "
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PC- 'as co(plete a #*;? agarose (idi gel
in 19 %AE buffer 'ith "8? S&berSafe
added 'as cast* 18 ul of each PC- reaction
put into a ne' tube and (ixed 'ith < ul of
39 sa(ple loading buffer the 1;ul sa(ples
'ere loaded into the 'ells 1/8 of the gel
'ith 1# ul of 1 Kb (ar.er in lane 3* %he gel
po'er suppl& 'as run at !# ,olts for
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had 1##ul of the negati,e control* All the
plates 'ere incubated for 1"/13 hours at
nce the
incubation of the gel 'as co(plete the gel
'as rinsed and i(aged*Purification and Protein Analysis of &is%
tagged $sgA
%he ti(e induction pellet 'ith the best
expression of the KsgA protein seen fro(the SDS/PA0E gel 'as tha'ed and had 8(l
of L&sis buffer added to it #*" (l of thesa(ple 'as sa,ed for protein deter(ination
and labeled starting cell pelletF* %he sa(ple
'as then sonicated for $8 seconds on and