α-complementation

• Occurs when two inactive fragments (α-acceptor and α-donor respectively) of E. coli β-galactosidase associate to form a functional enzyme.
• The α-acceptor fragment is generated by deletion of 5’ region of the lacZ gene which encodes the initiating methionine residues. This causes translation to begin at a downstream methionine residue and this generates a carboxy-terminal fragment of the enzyme.
• On the other hand, the α-donor fragment is the amino-terminal fragment generated by deletion or chain-terminal mutations in the structural gene.

Structure of β-galactosidase

• β-galactosidase is a tetramer composed of four identical monomers of 1023 amino acids each, which are folded into five sequential domains.
• The amino-terminal segment of the monomer is called the complementation region.

β-galactosidase in plasmid vectors

• Many plasmid vectors carry a short segment of E. coli DNA containing the regulatory sequences and coding information for the first 146 amino acids of the β-galactosidase gene.
• A polycloning site is embedded in the coding region which maintains the reading frame.
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  α-complementation

  This type of vectors is commonly used in host cells which express the carboxy-terminal portion of β-galactosidase.
• Although neither the host-encoded nor the plasmid-encoded fragments of β-galactosidase are active by themselves, they can associate to form an enzymatically active protein.
• This type of complementation, in which deletion mutants of the operator-proximal segment of the lacZ gene are complemented by β-galactosidase negative mutants that have the operator-proximal gene intact, is called α-complementation.
• The lac⁺ bacteria that result from α-complementation are easily recognized because they form blue colonies in the presence of the chromogen X-Gal.

Identification of recombinants using α-complementation

• Insertion of foreign DNA into the polycloning site of the plasmid invariably results in production of an amino-terminal fragment that is no longer capable of α-complementation à the bacteria carrying the insert form white colonies thus simplifying the identification of recombinants constructed in plasmid vectors.

Special Note:-

Screening by α-complementation is highly dependable but not completely infallible. Here’s why:

1. Insertion of foreign DNA does not always inactivate the complementing activity of the α-fragment of β-galactosidase. This happens mainly due to three reasons:
1. If the foreign DNA is small (<100 bp)
2. If the insertion does not disrupt the reading frame
3. If the insertion does not affect the structure of the α-fragment
2. Not all white colonies carry recombinant plasmids. Plasmids may be purged of their ability to express the α-fragment either due to mutation or loss of lac sequences. But the frequency of these lac- mutants is far lower than the number of lac⁺ generated in a ligation reaction.

IPTG

• Actual name: isopropyl-β-thiogalactoside
• Is a non-fermentable analog of lactose
• Inactivates the lacZ repressor thereby inducing transcription of the lac operon

X-Gal

• Actual name: 5-bromo-4-chloro-3-indolyl-β-D-galactoside
• Converted by β-galactosidase into an insoluble dense blue compound
• is sensitive and non-toxic to the bacteria it is used in

Why use β-galactosidase?

• It catalyzes two enzymatic reactions:
1. Hydrolysis of β-D-galactopyranosides. Example à hydrolysis of the disaccharide lactose into glucose and galactose
2. A transgalactosidation reaction in which lactose is converted into allolactose. Allolactose is the true inducer of the lac operon
• It in interacts with a series of synthetic analogs of lactose (in which glucose is replaced with other moieties). Some are chromogenic substrates such as:
• ONPG (ο-nitrophenyl-β-D-galactoside)
• X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside)
• MUG (4-methylumbelliferyl-β-D-galactoside)
• while others are inhibitors such as TPEG (p-aminophenyl-β-D-galactoside); it is used for affinity purification of lacZ fusion proteins.
• It is tolerant of deletions and substitutions of amino acids at its amino- and carboxyl- termini. For instance, up to 26 amino acids can be removed from the amino-terminus and replaced with several hundred more residues of a variety of other proteins without affecting enzyme activity.
• Is peculiar in that the amino- and carboxyl- domains need not be carried on the same molecule to generate β-galactosidase activity. Instead, two inactive fragments of the polypeptide chain à one lacking the amino-terminal region (α-donor) and the other the carboxy-terminal region (α-acceptor) are able to associate both in vivo and in vitro to form a tetrameric active enzyme…THE BASIS FOR α-COMPLEMENTATION!!!