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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