2) Protein Purification

what are the considerations of protein purification

source of protein

pH (avoid extreme pH values)

expression system (bacterial is simplest (consider gram+ or gram-)

tags (affinity tags)

pI of protein of fusion construct

cleavage of tag iwth protease

lysis conditions

second affinity chromatography step

how to get rid of fusion tag once it is cleaved

what are the most common separation methods?

IMAC: immobilized metal affinity chromatography

IEC: ion-exchange chromatography (anion or cation)

SEC: size exclusion chromatography

what are considerations with source of protein?

endogenous vs recombinant
endogenous = protein coming from native tissues/cells
recombinant = protein is over-expressed in expression system

tissue lysates vs expression systems
purifying from tissue lysates requires removing many other cellular proteins
recombinant expression systems may allow for simpler purification using engineered tags

what are types of expression systems? and details on them?

bacterial: (e coli) common for rapid expression and cost-effectiveness, but some proteins (eukaryotic), may not fold correctly or undergo proper post-translational modifications (PTMs) (look at gram+ or gram-)

yeast: (pichia pastoris), allows for some eukaryotic PTMs like glycosylation, but is still relatively fast and cost-effective. yeast glycosylation is eukaryotic but not necessarily human-like

insect cells: (baculovirus) offers a more complex environment for PTMs and protein folding, typically used for expressing proteins with more complex folding requirements

mammalian cells: (HEK293, CHO) ideal for proteins requiring human-like PTMs, but more expensive and slower than bacterial or yeast systems

what does gram + or gram - mean

gram+ have thick wall made of sugar and protein (peptidoglycan), and no outer membrane (staph and strep)

gram- have a thin wall of peptidoglycan hidden under extra outer protective layer (e coli and salmonella)

what are the most common types of tags?

what are

maltose binding

glutathione-s-transferase

6xHis

what is maltose binding protein (MBP)?

enhances solubility and is used in fusion with protein that tend to aggregate

binds to amylose/maltose for affinity purification

increases expression, used as an affinity tag

does not necessarily mean the target is correctly folded

what is glutathione-s-transferase (GST)?

solubility enhancing tag that binds glutathione (GSH) for affinity purification

dimeric, and can influence apparent oligomerization

what is a 6xHis tag?

short polyhistidine tag that binds to nickel, cobalt, and zinc resins

highly versatile and commonly used due to its small size and ease of use