×

"Good enzymes to prep in the lab instead of buying" thread posted on r/labrats subreddit taught me several fun facts. My doctoral training had me engrossed in recombinant protein design, expression, and purification. A well-behaved recombinant construct can yield a huge amount of protein that could net tens of thousands of dollars, if/when commercially sold^[1]. The advent of Addgene plasmid repository renders the field more accessible; for highly sought-after recombinant proteins, someone might have shared it. Case in point was SARS-CoV-2 spike glycoprotein with 6 stabilizing proline substitutions, dubbed as the S HexaPro shared by Dr. Jason McLellan's group. This was a critical reagent when I was completing my dissertation project on B cell response to SARS-CoV-2; S HexaPro is more stable, expressed at a higher yield, and more antigenic than the two-proline substitution version.

To quickly summarize the thread, here are the good targets:

• DNA polymerases in general.
  • Taq polymerase available on Addgene as pAKTaq #25712.
  • Phusion-family of DNA polymerases also available; see pOpen-P45 #165570, pATetO 6XHis-Phusion #206210, and pET16B.Pfu #12509.
  • Pfu-Sso7d polymerase construct design and expression protocol is available here, see notes by Pipette Jockey.
• HRV3C protease, frequently used for removing affinity purification tags from a recombinantly-expressed proteins.
  • Available on Addgene; see pHN924 #178885 and pET-NT*-HRV3CP #162795.
  • I prefer HRV3C because it is significantly faster than TEV.
  • Of note, cleaving His-tag from a recombinant antigen before immunizing is a good idea. His-tag is not completely immunogenically inert^[2].
• T7 RNA polymerase, an important tool for in vitro transcription studies.
  • See Addgene p6XHis-T7(P266L) #174866 and pCMV-T7 RNA POL #138352.
• T5 transposase for DNA fragmentation and tagging (tagmentation) for DNA library preparation (part of sequencing protocol).
  • See Addgene pTXB1-Tn5 #60240.
• Reverse transcriptase, a really really important reagent for a wide variety of basic research as well as for diagnostic laboratories.
  • Available on Addgene; see pET-19b_MMLV-RT #153312, pET_RTX #102787, and pOpen-MMLV_RT (lack H) #165556.
  • Unsure if this MMLV-RT is also the one for template-switching RT^[3].
  • The RTX is described to have a high fidelity, thanks to its proofreading activity.
• Biotin ligase (BirA from E. coli), a useful reagent for site-specific biotinylation of recombinant protein.
  • See Addgene pET21a-BirA #20857.
• T4 DNA Ligase, a critical reagent for routine restriction/digestion-based cloning and also for SLIC.
  • I could not find the plasmid, but methods are covered here: Chemically Incompetent's write-up on homebrew p50-T4 DNA ligase from 2019.
  • A paper by Robert H. Wilson et al. (PEDS 2013) "Engineered DNA ligases with improved activities in vitro"

A Case for Homebrewing Proteins

As of 2024/DEC/26, gold was sold at $84.91 per gram, or $0.008491 per 100 µg. A recombinant hemagglutinin protein^[4] from the H1N1 PR8 strain costs $348 per 100 µg, a whopping 40,984x difference in price. Granted, you cannot trade recombinant hemagglutinins at stock exchanges, but that's a massive difference in cost.

Of note, "protein yields" is not a straightforward concept. A variety of expression systems exist, and bacterial expression (typically E. coli strain BL21) seems to be the favorite for expressing enzymes for molecular biology applications. Estimates vary (owing to the induction and culture conditions); a ballpark yield for Taq polymerase could reach approximately 5 mg of purified Taq per 1 liter of culture. Assuming a specific activity of 250,000 units per milligram, that adds up to 1,250,000 units of Taq produced. NEB sells Taq DNA polymerase for $1,398 for 20,000 units, which means our homebrew liquid gold could reach $80,000 – $100,000 from a single purification. Alex from Pipette Jockey did a similar calculation for homebrew Pfu-Sso7d and our estimates are in agreement.

Aside from enzymes, monoclonal antibodies are also the hot target. Using a mammalian expression system (Expi293 cell line^[5]), I successfully purified for CR9114 monoclonal antibody at a yield of 10 mg purified protein per 100 mL expression culture^[6]. I usually reach out for BioXCell as they sell monoclonals at a cheap, affordable, and fair price (e.g. InVivoMAb anti-human c-myc clone 9E10 BE0238 at $600 for 5 mg). Abcam, on the other hand, sells 9E10 monoclonal at $3,600 for 1 mg. If I were to follow in the footsteps of Abcam, I could have made $36,000 from selling CR9114 that I made with 100 mL of Expi293 culture. Now, imagine if I actually expressed the monoclonal in 1 liter of culture.

It would be terribly misleading if we just focus on the protein yields and the resulting monetary gain while excluding the cost to make them. Expi293 system is not cheap; the Expi293 expression medium (chemically-defined) costs $1,874 for 6 liters. PEI+VPA is the cheap alternative to expressing protein in mammalian system, but it is not nothing. Including the purification cost (and the expertise needed), this is where it becomes limiting for a lot of labs. Nickel-based purification of His-tagged protein remains the most economical purification method at $6.80 per purification, while protein G for monoclonal antibodies would cost $50.94 per purification^[7]. See below for the cost per purification when only factoring for the resin (price here as of March 2024).

Protein Expression Joint Laboratory Core

I consider coding/programming as one of the greatest modern arts of wizardry. Protein design/expression/purification is either tied (when it works) with coding, or comes close at the second (when I have to troubleshoot). It is an exercise in owning your production line and to offshore expertise as little as possible. Of many lessons I learned in graduate school, the ones that did not make into my doctoral dissertation were as useful. Pertinent to this topic is protein production specifically, and "I can be my own core facility" generally. ELISA and ELISpot already demanded a huge amount of antigens per project, and that was before factoring in for flow cytometry assays that required a fluorescently-labeled B-cell antigen tetramer probes.

That being said, this exercise could be a demanding one; a time hog and resource hog. Each protein lot must be validated, and often in multiple orthogonal assays to ensure quality. From design to purification and to validation, this could consume 1.5 – 2 months of work, assuming from scratch. Not everybody has to do it, but everyone should experience it at least once to appreciate the craft. I do think there is a huge benefit for a research department to have a core laboratory that can assist with design, expression, and production of high demand proteins. Commonly-used monoclonal antibodies for routine detections and highly in-demand enzymes such as Pfu-Sso7d & MMLV-RT could already save tens of thousands in reagent cost. Owning our own production could be cheaper than we think.