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Title: Generation and Use of Recombinant Galectins
Abstract

Galectins are soluble carbohydrate binding proteins that can bind β‐galactose‐containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host‐pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan‐binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl‐sepharose and nickel‐NTA affinity chromatography using human galectin‐4N and ‐7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin‐1 and ‐9 as examples. © 2021 Wiley Periodicals LLC.

This article was corrected on 20 July 2022. See the end of the full text for details.

Basic Protocol 1: Purification of galectins using lactosyl‐sepharose affinity chromatography

Basic Protocol 2: Purification of human galectin‐7 using a nickel‐NTA affinity chromatography column

Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin‐1

Alternate Protocol 2: Purification of human galectin‐9 using lactosyl‐sepharose column chromatography

 
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NSF-PAR ID:
10226680
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
1
Issue:
3
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time‐saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC.

    Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour

    Alternate Protocol 1: Preparation of crude extracts from Jack bean meal

    Alternate Protocol 2: Preparation of crude extracts from the corms ofColocasia esculenta,Xanthosoma sagittifolium, and from the bulbs ofAllium sativum

    Alternate Protocol 3: Preparation ofEscherichia colicell lysates containing human galectin‐3

    Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin)

    Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension

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    Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target‐TCA complex from other components of the crude extracts

    Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex

    Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents

    Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins)

     
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    Basic Protocol 1: Lentivirus production and expression line creation

    Support Protocol 1: Six‐well assay for estimation of production cell line yield

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    Methods

    A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed inEscherichia coliwere immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.

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    Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarifiedE. colicell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.

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